Acyl glucosaminyl inositol amidase family and methods of use

ABSTRACT

The present invention provides a family of bacterial acyl glucosaminylinositol amidases with amidase activity against S-conjugate amides, particularly mycothiol-derived S-conjugate amides. The invention amidases are characterized by a highly conserved 20 amino acid N-terminal region and four highly conserved histidine-containing regions and by having amidase activity, particularly amide hydrolase activity. The invention further provides methods for using the invention amidases in drug screening assays to determine compounds with antibiotic activity or compounds that inhibit activity or production of endogenous acyl glucosaminyl inositol amidase in bacteria. The invention further provides methods for detoxifying a toxic substance by contacting the toxic substance with an invention amidase, for example, by expression of the amidase under environmental conditions in a bacterium.

RELATED APPLICATION

[0001] This application relies for priority upon U.S. Provisionalapplication Serial No. 60/169,503, filed Dec. 7, 1999.

FIELD OF THE INVENTION

[0002] The present invention generally relates to a family of enzymaticcompounds produced by bacteria and methods of their use in drugdiscovery and degradation of toxic substances, and more specifically toacyl glucosaminyl inositol amidases and methods of their use.

BACKGROUND OF THE INVENTION

[0003] Aerobic organisms are subjected to oxidative stress from manysources, including atmospheric oxygen, basal metabolic activities, and,in the case of pathogenic microorganisms, toxic oxidants from the hostphagocytic response intended to destroy the bacterial invader.Glutathione (GSH) is the dominant low molecular weight thiol in mosteukaryotes and Gram-negative bacteria, and it plays a key role inprotection of the cell against oxygen toxicity and electrophilic toxins(R. C. Fahey and A. R. Sundquist (1991) Adv. Enzymol. 64:1-53; Dolphin,et al, (1989) Glutathione: Chemical, Biochemical, and Medical Aspects pp45-84, John Wiley & Sons, New York). However, actinomycetes, includingStreptomyces and Mycobacteria do not make GSH but produce millimolarlevels of mycothiol (MSH, AcCys-GlcN-Ins), an unusual conjugate ofN-acetylcysteine (AcCys) with1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) (G. L.Newton, et al. (1996) J. Bacteriol. 178:1990-1995; S. Sakuda, et al.,(1994) Biosci. Biotech. Biochem. 58:1347-1348; H. S. C. Spies and D. J.Steenkamp, (1994) Eur. J. Biochem. 224:203-213; G. L. Newton, et al.(1995) Eur. J Biochem. 230:821-825) (FIG. 1A).

[0004] Mycothiol autoxidizes more slowly than glutathione (G. L. Newton,et al. (1995) Eur. J. Biochem. 230:821-825) and mutants of Mycobacteriumsmegmatis defective in the biosynthesis of mycothiol have increasedsensitivity to hydrogen peroxide and antibiotics relative to the parentstrain (G. L. Newton, et al. (1999) Biochem. Biophys. Res. Commun.255:239-244). This observation suggests that mycothiol may play a keyrole in the protection of actinomycetes against oxygen toxicity andreactive toxins. The biochemistry of mycothiol appears to have evolvedcompletely independently of that of glutathione.

[0005] However, it has already been established that the metabolism ofmycothiol parallels that of glutathione metabolism in two enzymaticprocesses. First, formaldehyde is detoxified in glutathione-producingorganisms by NAD/glutathione-dependent formaldehyde dehydrogenase (L.Uotila, et al. (1989) in Glutathione: Chemical, Biochemical, and MedicalAspects—Part A (D. Dolphin, et al., Eds.) pp 517-551, John Wiley & Sons,et al.). An analogous process involving NAD/mycothiol-dependentformaldehyde dehydrogenase has been identified in the actinomyceteAmycolatopsis methanolica (M. Misset-Smits, et al. (1997) FEBS Lett.409:221-222). This enzyme has been sequenced (A. Norin, et al. (1997)Eur. J. Biochem. 248:282-289).

[0006] A mycothiol homolog of glutathione reductase was recently clonedfrom M. tuberculosis and expressed in M. smegmatis (M. P. Patel, et al.(1999) J. Amer. Chem. Soc. 120:11538-11539, M. P. Patel, et al. (1999)Biochem. 38:11827-11833). The reductase is reasonably specific for thedisulfide of mycothiol but is also active with the disulfide ofAcCys-GlcN, the desmyo-inositol derivative of mycothiol (M. P. Patel, etal. (1999) supra.). Therefore, there is a need in the art forinvestigation of the details of the metabolism of mycothiol andcomparison with the established roles for the metabolism of glutathione.

[0007] Antibiotic resistance of pathogenic bacteria, includingpathogenic actinomycetes, such as M. tuberculosis, is a well-knownproblem faced by medical practitioners in treatment of bacterialdiseases. Therefore, there is a further need in the art for screeningtechniques to discover new antibiotics and drugs effective to reduceresistance to existing antibiotics in treatment of bacterial infectionsin humans and in other mammals, such as domestic and farm animals.

[0008] Air, soil and groundwater in areas surrounding industrial centersand farming areas are becoming increasingly polluted with simple organiccompounds with have long lifetimes in the environment. These compoundsinclude, but are not limited to 1, 2 dibromoethane, 1,2 dichloroethane,perchloroethene, trichloroethene, isoprene, and vinyl chloride. They arefrom pesticides, industrial degreasers, solvents, and from theproduction polyvinyl chloride polymers (plastics). Organisms haverecently been isolated from contaminated environments that have theability to detoxify, and in some cases grow using these pollutants as asole carbon source. There is great interest in industrialized countriesin using microorganisms for biodegradation of these pollutants in soiland groundwater, a field generally known as bioremediation.

[0009] Recent reports indicate that vinyl chloride, 1,2 dibromoethane,and numerous other haloalkanes are detoxified by mycobacteria (A.Jesenke et al., Microbiology, 66:2219-222 (2000); S. Hartmans, and A. M.DeBont, Applied and Environmental Microbiology, 58:1220-1226 (1992).; G.J. Poelarends, et al. J. Bacteriol., 181:2050-2058, (1999)). These toxiccompounds are generally dehalogenated to form epoxides ormonohaloaldehydes that are in turn toxic compounds to microorganismsuntil they are conjugated with thiols. Many of these organisms areactinomycetes and are likely to have mycothiol and mycothiolbiosynthesis (G. L. Newton, et al. (1996) J. Bacteriol., 178:1990-1995).In the case of mycobacteria, mycothiol is the major low molecular weightthiol and will form a mycothiol conjugate. The product of thisconjugation may still be toxic. Although such studies have shown theneed for low molecular weight thiols in the detoxification reactions fortoxins and have assayed their organisms for glutathione, to date suchstudies have not acknowledged the occurrence of mycothiol inmycobacteria.

[0010] Another actinomycete, Rhodococcus sp. Strain AD45 has beenextensively studied for detoxification of isoprene, 1,2 dibromoethaneand 1,2 dichloroethene (J. E. T. van Hylckama Vlieg, et al., CurrentOpinion in Microbiology, 3:257-262 (2000)). The enzymes responsible forthe detoxification of these toxic substances were claimed to include aglutathione S-transferase and a glutathione conjugate specificdehydrogenase (J. E. T. van Hylckama Vlieg, et al, Applied andEnvironmental Microbiology, 64:2800-2805 (1998); J. E. T. van HylckamaVlieg, et al. J. Bacteriology, 181:2094-2101 (1999); J. E. T. VanHylckama Vlieg et al., J. Bacteriology 182:1956-1963 (2000).

[0011] Thus, there is a further need in the art for methods andcompounds useful for detoxification of environmental toxins.

SUMMARY OF THE INVENTION

[0012] The present invention overcomes these and other problems in theart by providing a family of purified acyl glucosaminyl inositol amidasepolypeptides with enzymatic amidase activity for glucosaminyl inositol(GlcN-Ins)-containing substrates. The invention acyl glucosaminylinositol amidases are characterized by comprising an N-terminal regionwith an amino acid sequence with at least 80% sequence identity to SEQID NO:2, having four highly conserved domains wherein three of thedomains contain conserved histidine residues, and having amidaseactivity against glucosaminyl inositol-containing amides.

[0013] In another embodiment according to the present invention, thereare provided methods for identifying an inhibitor of acyl glucosaminylinositol amidase activity by contacting a candidate inhibitor with anacyl glucosaminyl inositol amidase or a polynucleotide encoding theamidase in the presence of an GlcN-Ins-containing amide under suitableconditions and then determining the presence or absence of breakdownproducts of the amide indicative of amide hydrolase activity. Thesubstantial absence of the amide hydrolase activity indicates thecandidate compound is an inhibitor of acyl glucosaminyl inositol amidaseactivity.

[0014] In still another embodiment according to the present invention,there are provided methods for increasing production of antibiotic byantibiotic-producing bacteria by contacting the antibiotic-producingbacteria with a compound that increases intracellular production by thebacteria in culture of an acyl glucosaminyl inositol amidase. Theincrease in production of the amidase increases the production ofantibiotic by the bacteria by increasing resistance of the bacteria tothe antibiotic.

[0015] In yet another embodiment according to the present invention,there are provided methods for decreasing the antibiotic-resistance ofpathogenic acyl glucosaminyl inositol amidase-producing bacteria byintroducing into the bacteria an inhibitor of acyl glucosaminyl inositolamidase activity. The intracellular presence of the inhibitor decreasesactivity of the amidase, thereby decreasing the antibiotic-resistance ofthe bacteria as compared with untreated control bacteria.

[0016] In yet another embodiment according to the present invention,there are provided methods for detoxifying a toxic substance comprisingcontacting the toxic substance with bacteria transformed with apolynucleotide that encodes an acyl glucosaminyl inositol amidase andexpressing the amidase in order to detoxify the toxic substance.

[0017] In still another embodiment according to the present invention,there are provided processes for preparation of GlcN-Ins by contactingan N-acyl glucosaminyl inositol under suitable conditions with an acylglucosaminyl inositol amidase so as to therein obtain the GlcN-Ins.

BRIEF DESCRIPTION OF THE FIGURES

[0018]FIG. 1A is a drawing showing the chemical structure of mycothiol(AcCys-GlcN-Ins) (MSH), chemical name1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside.

[0019]FIG. 1B is a schematic representation of the reaction wherein MSHis alkylated (R=alkylating group) to MSR and broken down by enzymemycothiol S-conjugate amidase (MCA) to form1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) and amercapturic acid (AcCysR).

[0020]FIG. 1C is a schematic representation showing hydrolysis of bimanederivative (MSmB) formed by alkylation of mycothiol by monobromobimane(mBBr), which is cleaved to produce GlcN-Ins and the bimane derivativeof N-acetylcysteine (AcCySmB), a mercapturic acid.

[0021]FIG. 2 is a schematic representation of vector pYA1082E.

[0022]FIG. 3 is a graph showing detoxification of mBBr by exponentiallygrowing cells of M. smegmatis. AcCySmB content of cells=open circle;AcCySmB content of medium=dark circle; cellular content of MSH=opensquare; cellular content of GlcN-Ins=open triangle.

[0023]FIG. 4 is a graph showing the final step in purification of M.smegmatis amidase from Sephadex G-100 chromatography of highest specificactivity fractions from Phenyl-Sepharose chromatography. A280=absorbance280 nm, dark circles; amidase activity=open circles.

[0024]FIG. 5 is a schematic drawing showing MSH-dependent detoxificationof mBBr or other toxins by mycobacteria.

[0025]FIG. 6 is a chart showing alignment of the amino acid sequences offive homologs of M. smegmatis mycothiol S-conjugate amidase designatedby the genes that encode them: Rv1082=the amidase gene from M.tuberculosis H37Rv ; 1mbE=the lincomycin biosynthesis gene E fromStreptomyces lincolnensis; rifO=rifamycin biosynthesis gene O fromAmycolaptosis mediterranei; Rv1170=an acetyl glucosaminyl inositol(N-Acetyl-1-D-myo-Inosityl-2 amino-2 deoxy-α-D-glucopyranoside)deacetylase from M. tuberculosis H37Rv (G. L. Newton et al., J Bacterol.182(24):6958-6963 (2000).

[0026]FIG. 7 is a chart showing chemical structures of varioussubstrates for mycothiol S-conjugate amidase with changes in thestructure of the mycothiol moiety. MSmB activity is defined as 100%.

[0027]FIG. 8 is a chart showing the chemical structures of variousS-conjugate substrates used to test the substrate-specificity ofmycothiol S-conjugate amidase as shown in Table 2 herein. The relativeactivities compared with MSmB taken as 100% are shown in parentheses.

[0028]FIG. 9 is a chart showing alignment of the amino acid sequences ofthe mycothiol S-conjugate amidases of M. smegmatis mc2 155 (SEQ ID NO:1)(source TIGR) and M. tuberculosis H37Rv (Rv1082) (SEQ ID:7) (SourceSanger Center). The two sequences are 78% identical overall and theN-terminal 20 amino acids are 100% identical (SEQ ID:8).

[0029]FIG. 10 shows the nucleic acid sequences encoding the mycothiolS-conjugate amidase of M. smegmatis (SEQ ID NO:6) and M. tuberculosis(SEQ ID:9)

DETAILED DESCRIPTION OF THE INVENTION

[0030] In accordance with the present invention, there are provided afamily of purified acyl glucosaminyl inositol amidase polypeptides withenzymatic amidase activity for glucosaminyl inositol(GlcN-Ins)-containing substrates. The invention acyl glucosaminylinositol amidases are characterized by having an N-terminal region withan amino acid sequence with at least 80% sequence identity to amino acidsequence MSELRLMAVHAHPDDESSKG (SEQ ID NO:2), four highly conserveddomains wherein three of the domains contain conserved histidineresidues, and amidase activity against glucosaminyl inositol-containingamides.

[0031] In preferred embodiments the invention polypeptides have enzymeactivity as an amide hydrolase and the three histidine-containingconserved regions are selected from V/F-HAHPDD (SEQ ID NO:3) of domain1, D/HPDHINV (SEQ ID NO:4) of domain 3, and ALX-A/S-H-A/V-T/S-Q (SEQ IDNO:5) of domain 4 as shown in FIG. 6 and FIG. 9, or any combination ofany two or more thereof.

[0032] A subset of the acyl glucosaminyl inositol amidases are referredto herein as S-conjugate amidases, whose substrate is an S-conjugatecontaining amide. As used herein, the term “S-conjugate” means that themolecule is a thioether or thioester containing two chemical moietiesjoined by a sulfur (i.e., —S—) moiety. In a preferred embodiment theS-conjugate molecule is derived from mycothiol (FIG. 1A) by the reactionshown in FIG. 1B, wherein RX is an electrophile and R is an alkyl oralkyloid moiety. However, the acyl glucosaminyl inositol amidases of theinvention acyl glucosaminyl inositol amidase family do not require asulfur-containing amide substrate and instead cleave anGlcN-Ins-containing amide substrate.

[0033] As used herein, the terms “GlcN-Ins-containing amide” and“glucosaminyl inositol-containing amide” are interchangeable when usedto describe a substrate molecule for which a member of the inventionfamily of amidases have enzymatic activity, resulting in cleavage of themolecule. Similarly, the term “amide-containing S-conjugate” and“S-conjugate-containing amide” are interchangeable when used to describea substrate molecule for which a member of the invention S-conjugateamidases have enzymatic activity, resulting in cleavage of the molecule.If a particular member of the invention family of polypeptide amidasesis an amide hydrolase, cleavage of the substrate molecule will formbreakdown products wherein one product is a carboxylic acid, (e.g., acarboxylic acid containin at least one sulfur moiety) and the otherproduct is a amine (e.g., GlcN-Ins). If the substrate is amycothiol-derived S-conjugate amide of the type illustrated in FIG. 1B,one of the breakdown products will be1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) and theother breakdown product will be a sulfur-containing carboxylic acid,such as a mercapturic acid. AcCys S-conjugates are termed mercapturicacids, the final excreted product in the mercapturic acid pathway ofglutathione-dependent detoxification in mammals (J. L. Stevens, et al.,(1989) in Glutathione: Chemical, Biochemical, and Medical Aspects—Part B(D. Dolphin, et al.) pp 45-84, John Wiley & Sons, et al.).

[0034] It has been discovered that invention acyl glucosaminyl inositolamidases participate in a pathway of detoxification in bacteria,especially antibiotic-producing bacteria, and that the detoxificationpathway is dependent on in vivo production of a protein acylglucosaminyl inositol amidase by such bacteria. However, pathogenicactinomycetes (that do not produce an antibiotic) also contain a geneencoding an acyl glucosaminyl inositol amidase that becomes activated inthe presence of antibiotics administered to a host, for example intreatment of a disease caused by the pathogenic actinomycetes. Thus, thegene(s) encoding the invention family of amidases are a family ofantibiotic-resistance genes.

[0035] More particularly, it has been discovered that mycothiol(1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside)(MSH) is present in a variety of actinomycetes and plays an essentialrole in a pathway of detoxification in such bacteria. Mycothiol iscomprised of N-acetylcysteine (AcCys) amide linked to1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins) and isthe major thiol produced by most actinomycetes. In themycothiol-dependent detoxification process in actinomycetes, analkylating agent is converted to a S-conjugate of mycothiol, the latteris cleaved to release a mercapturic acid, and the mercapturic acid isexcreted from the cell. This process has similarities to the mercapturicacid pathway for glutathione-dependent detoxification in highereukaryotes (J. L. Stevens, et al. (1989) supra.) but involves fewersteps.

[0036] An S-conjugate amidase responsible for cleavage of theS-conjugate of mycothiol has been purified from M. smegmatis (SEQ IDNO:1 shown in FIG. 9) and was found to be located at amino acid residues5717 through 4858 of a plasmid having Sanger Center Accession No.GMS-684. The N-terminal region 20 residues of this newly discoveredS-conjugate amidase was determined as shown in SEQ ID NO:2. The nucleicacid sequence that encodes the M. smegmatis S-conjugate amidase (SEQ IDNO:6) is found at nucleic acid residues 3854 to 6717 of the plasmidhaving Sanger Center Accession No. GMS-684, shown in FIG. 10.

[0037] An open reading frame encoding an identical predictedamino-terminal amino acid sequence was also identified in the Mtuberculosis genome (FIG. 9). The Rv1082 gene (mca) from M. tuberculosiswas inserted into vector pYA1082E (FIG. 2) and expressed in E. coli, andthe expressed protein was shown to have substrate specificity similar tothe invention amidase from M smegmatis. These results indicate thatmycothiol and mycothiol S-conjugate amidases play an important role inthe detoxification of alkylating agents and antibiotics.

[0038] When M. tuberculosis S-conjugate amidase was used to searchgenomic databases, few proteins with similar size were identified asclose homologs. Interestingly, all identified homologous proteins wereputative with heretofore unknown function. As expected from theenzymatic activity utilizing mycothiol-S-conjugates as substrates for M.smegmatis and M. tuberculosis-derived amidases, most of the homologousnon-mycobacterial proteins were found in actinomycetes which producemycothiol. Homolog genes encoding S-conjugate amidases were found to belocated within antibiotic synthesis operons of the antibiotic producersStreptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsisorientalis, Streptomyces lavendulae, Streptomyces coelicolor,Streptomyces rochei, and the polyketide erythromycin antibiotic producerSaccharopolyspora erythraea.

[0039] The sequence alignment also provides information that was foundby screening for S-conjugate amidase homologs against bacterial genomicsequence databases. Within M. tuberculosis an open reading frame, whichencodes for homolog Rv1170, was identified. In other mycobacteria, suchas M. leprae, a member of invention family of acyl glucosaminyl inositolamidases is encoded by ORF 05988 located in the cosmid B1740, and the M.avium homolog was represented in a contig 9 in the TIGR genomedatabases. An additional S-conjugate amidase homolog was also identifiedin the M. bovis genome database that is currently underway at the SangerCentre. Interestingly two other bacteria, Corynebacterium diphtheria andDeinococcus radiodurans, were also found to encode acyl glucosaminylinositol amidase homologs. Thus, protein homologs in GenBank (NationalCenter for Biotechnology Information, Building 38A, Room 8N805,Bethesda, Md. 20894) for which function had not previously beenidentified were identified as members of the invention family of acylglucosaminyl inositol amidases by comparative analysis. Four of theseproteins are putative actinomycetes proteins encoded within thelincomycin, erythromycin, mitomycin and the rifamycin antibioticbiosynthetic operons.

[0040] To identify functional domains within the group of homologs, asearch for known protein motifs or domains was performed against proteindatabases. No known functional domains were identified. However,ClustalW alignment (See FIG. 6) of M. tuberculosis mycothiol S-conjugateamidase against several homologs, revealed at least four major domainsthat are highly conserved among members of the invention family ofpolypeptide amidases. Three out of the four domains contain conservedhistidine residues: V/F-HAHPDD (SEQ ID NO:3) of domain 1, D/HPDHI/V (SEQID NO:4) of domain 3, and ALX-A/S-H-A/V-T/S-Q (SEQ ID NO:5) of domain 4.These conserved domains are thought to be involved in the amidehydrolysis and binding to glucosaminyl inositol. Furthermore, thisalignment clearly demonstrates that members of invention family of acylglucosaminyl inositol amidase proteins are highly conserved and share ahigh degree of identity throughout the whole protein. Those of skill inthe art will be able to identify additional members of the acylglucosaminyl inositol amidase family by performing similar homologysearches and sequence alignments using available genomic databases, andthe like, in conjunction with any of a number of sequence alignmentprograms commercially available. Genes coding for mycothiol S-conjugateamidase appear to be present in all of the mycobacterial genomespresently available and it seems likely that homologs will be found inother mycothiol-producing actinomycetes.

[0041] Members of invention family of acyl glucosaminyl inositolamidases are formed in vivo by bacteria as part of a detoxificationpathway, usually in antibiotic-producing bacteria, and most usually inbacteria characterized by intracellular production of mycothiol. WhenMycobacterium smegmatis was treated with the alkylating agentmonobromobimane (mBBr), the cellular mycothiol was converted to itsbimane derivative (MSmB) (FIG. 1C). The latter was rapidly cleaved toproduce GlcN-Ins and the bimane derivative of N-acetylcysteine(AcCySmB), a mercapturic acid that was rapidly exported from the cellsinto the medium. The other product of cleavage, GlcN-Ins, was retainedin the cell and utilized in the resynthesis of mycothiol. This reactionscheme is shown in FIG. 5.

[0042] The substrate specificity of the amidase reaction was examinedusing various mycothiol related compounds (Table 2; FIG. 7). A very lowbut measurable activity was found with mycothiol under these conditions.When mycothiol was examined at 2 mM, a concentration comparable to thecellular level of mycothiol (S. J. Anderberg, et al. (1998) supra.),under conditions otherwise the same as for Table 2 the rate of GlcN-Insformation was 14±1 nmol/min/mg, or 0.3% of the rate with 0.1 mM MSmB.Mycothiol disulfide was a better substrate than mycothiol but still <1%as reactive as MSmB. Removal of the acetyl group from MSmB resulted in a10³-fold reduction in rate and removal of the inositol residue produceda >10⁴-fold loss of activity (FIG. 7). The invention amidase exhibitedsubstantial activity with a wide range of S-conjugates other than MSmB,including the S-conjugate of the antibiotic cerulenin (S. Omura (1981)Meth. Enzymol. 72:520-532) which serves as an example of a naturallyoccurring substrate (Table 2, FIG. 8).

[0043] Tests were also conducted to determine whether mycothiol inhibitsthe cleavage of 100 μM MSmB by invention S-conjugate amidase. Theamidase activity was decreased by 30±3, 48±3, and 89±3% (n=2) at 1, 3,and 10 mM mycothiol, respectively. This suggests that cellular levels ofmycothiol could produce significant inhibition of the monomeric amidase,the presumed form under these assay conditions.

[0044] Purified M smegmatis mycothiol S-conjugate amidase was assayedfor cysteine:GlcN-Ins ligase activity, a mycothiol biosynthesis enzyme(C. Bornemann, et al. (1997) supra.). The reaction utilizes ATP to driveformation of an amide bond of the type hydrolyzed by inventionS-conjugate amidase. The purified amidase was incubated with ATP,cysteine and GlcN-Ins, and the product, Cys-GlcN-Ins, was assayed byHPLC as the bimane derivative (S. J. Anderberg, et al. (1998) supra.).The purified amidase (0.044 μg) gave <0.33 nmol/min/mg Cys-GlcN-Ins at aprotein concentration where the amidase reaction rate for 30 μM MSmB was˜3000 nmol/min/mg. As a positive control the ligase reaction was alsoassayed for a dialyzed crude extract from M. smegmatis and 0.36nmol/min/mg protein Cys-GlcN-Ins was formed in accord with previousreports (Newton, et al. (1999), supra., S. J. Anderberg, et al. (1998)supra.). Thus, invention S-conjugate amidase does not appear to beinvolved in mycothiol biosynthesis since it has no significant abilityto catalyze ATP-dependent ligation of cysteine with GlcN-Ins. Ittherefore does not appear to be a bifunctional enzyme analogous to theglutathionylspermidine synthetase/amidase which catalyzes both thebiosynthesis and degradation of glutathionylspermidine in E. coli (D. S.Kwon, et al. (1997) J. Biol. Chem. 272:2429-2436) and in Crithidiafasciculata (E. Tetaud, et al. (1998) J. Biol. Chem. 273:19383-19390).Attempts to detect mycothiol S-transferase activity in extracts of Msmegmatis using 1-chloro-2,4-dinitrobenzene with 1 mM MSH andmonochlorobimane with 0.1 mM MSH did not produce significant activity(data not shown).

[0045]E. coli has no mycothiol metabolism and is not expected to containmycothiol conjugate amidase endogenous proteins that would givebackground to these assays. The amidase activity of the M.tuberculosis-derived S-conjugate amidase expressed in E. coli was foundto be associated with the insoluble cell pellet material. Using 0.1 mMMSmB as substrate, the resolublilzed crude protein extract was found toproduce 4.1±0.05 nmoles/min/mg protein GlcN-Ins and 5.4±0.3nmoles/min/mg protein AcCysmB. When using 0.1 mM MSH as substrate<0.0023 nmoles/min/mg protein GlcN-Ins was produced by the same extract.The products of the reaction and the >2,000 fold rate enhancement forconjugates of MSH relative to the free thiol is very similar toinvention S-conjugate amidase purified from M. smegmatis.

[0046] Based on these activity studies, which are described more fullyin the Examples hereinbelow, it is concluded that acyl glucosaminylinositol amidases (for example, S-conjugate) amidases participate indetoxification of antibiotics or the antibiotic biosynthesis by-productsin actinomycetes and other bacteria. In disease states characterized bythe presence of such pathogenic bacteria (e.g., bacterial infections),therapeutic antibiotics administered to the subject being treated mayhave limited effectiveness in treating the disease because of innateresistance of the pathogenic bacterium to antibiotics subject to such adetoxification pathway. Such a bacterium may prove resistant to thetherapeutic antibiotic administered to the subject hosting thebacterium. The studies described herein indicate that pathogenicbacteria particularly susceptible to such resistance are pathogenicactinomycetes, such as those derived from M. smegmatis, M. tuberculosis,M. leprae, M. bovis, Corynebacterium diphtheria, Actinomycetes israelii,M. avium, and the like, that can produce a native GlcN-Ins-containingamide.

[0047] Accordingly, in another embodiment of the present invention,there are provided methods for identifying an inhibitor of acylglucosaminyl inositol amidase activity by contacting a candidatecompound with an acyl glucosaminyl inositol amidase or a polynucleotideencoding the amidase in the presence of an GlcN-Ins-containing amideunder suitable conditions and then determining the presence or absenceof breakdown products of the amide indicative of amide hydrolaseactivity. The substantial absence of the amide hydrolase activity isindicative of a compound that inhibits activity of the amidase. Forexample, if the amidase is an S-conjugate amidase, the absence ofmercapturic acid (AcCysR) and/or GlcN-Ins as breakdown productsindicates the candidate compound is an inhibitor of the S-conjugateamidase. The inhibitor may be a polypeptide, oligonucleotide, or smallmolecule. When administered in treatment of a disease associated withinfection of the subject with a pathogenic bacteria that produces anative acyl glucosaminyl inositol amidase in conjunction with anantibiotic (i.e., in combination therapy) the inhibitor increases thetherapeutic effect of the antibiotic.

[0048] Preferably, the inhibitor is a compound, such as an antisenseoligonucleotide, that inhibits intracellular production of the amidase.For example, the antisense oligonucleotide can be complementary to atarget region in a messenger RNA that encodes a polypeptide having anamino acid sequence segment with at least 80% sequence identity to theamino acid sequence of SEQ ID NOS:2, 3, 4 or 5 and conservativevariations thereof. In another embodiment the antisense oligonucleotidehybridizes under intracellular conditions with a messenger RNA thatencodes a polypeptide having an N-terminal amino acid sequence as setforth in SEQ ID NO:2.

[0049] For example, in one embodiment, the candidate compound inhibitsintracellular production or activity of the acyl glucosaminyl inositolamidase. A presently preferred drug candidate for screening in livebacteria for activity that inhibits intracellular production or activityof acyl glucosaminyl inositol amidase is an anti-sense oligonucleotidecomplementary to a target region in a messenger RNA that encodes apolypeptide having an N-terminal amino acid sequence with at least 80%sequence identity to the amino acid sequence set forth in SEQ ID NO:2,or a conservative variation thereof, for example, 85%, 90%, 95% or 100%sequence identity. Suitable conditions for conducting invention drugscreening methods are well known in the art and are described, forexample, in the Examples hereinbelow.

[0050] In yet another embodiment according to the present invention,there are provided methods for decreasing the antibiotic-resistance ofpathogenic GlcN-Ins-amidase producing bacteria by introducing into thebacteria an inhibitor of acyl glucosaminyl inositol amidase activity.The intracellular presence of the inhibitor decreases activity of theamidase, thereby decreasing the antibiotic-resistance of the bacteria ascompared with untreated control bacteria. The inhibitor can be apolypeptide, polynucleotide or oligonucleotide, or a small moleculePreferably, the inhibitor inhibits intracellular production of theamidase.

[0051] For example, the inhibitor can be an antisense oligonucleotidecomplementary to a target region in a messenger RNA that encodes an acylglucosaminyl inositol amidase polypeptide that is introduced into thepathogenic GlcN-Ins-amidase producing bacteria, using methods known inthe art and as described herein. For example, the pathogenic bacteriacan be contacted with an antisense oligonucleotide that hybridizes underintra cellular conditions with a messenger RNA that encodes an aminoacid sequence segment with at least 80% sequence identity to the aminoacid sequence of SEQ ID NOS:2, 3, 4, or 5 and conservative variationsthereof or a polypeptide having an N-terminal amino acid sequence as setforth in SEQ ID NO:2.

[0052] Preferably, the pathogenic bacteria treated to reduce drugresistance according to the invention methods are actinomycetes, such asM. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare,M. africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria,Actinomycetes israelii, M. avium, and the like.

[0053] In one embodiment, the amidase produced by the pathogenicbacteria is mycothiol S-conjugate amidase, e.g. one capable ofhydrlyzing a mycothiol S-conjugate where the S-R group may be an alkylor alkyloid group.

[0054] In still another embodiment according to the present invention,there are provided methods for increasing production of antibiotic byantibiotic-producing bacteria by contacting the antibiotic-producingbacteria with a compound that increases intracellular production by thebacteria in culture of an acyl glucosaminyl inositol amidase. Theincrease in intracellular production of the amidase increases theproduction of antibiotic by the bacteria by increasing resistance of thebacteria to the antibiotic. Generally, in industrial applicationswherein antibiotic is produced from bacteria for commercial purposes,the antibiotic-producing bacteria are cultured under conditions suitablefor production of the antibiotic, and the antibiotic is recovered fromthe culture media. The compound that increases intracellular productionby the bacteria of the amidase can be a polypeptide, polynucleotide, orsmall molecule.

[0055] Preferably, the compound that increases intracellular productionby the bacteria of the amidase is expressed intracellularly by thebacteria, preferably by actinomycetes. For example, the actinomycetescan be transformed with a polynucleotide that encodes an acylglucosaminyl inositol and amidase and which expresses the amidase inculture. Recombinant expression of the acyl glucosaminyl inositolamidase polypeptides in cultured antibiotic-producing cells can beuseful for increasing the resistance of the production cells to thetoxic effect upon themselves of the antibiotics they produce. Thus, thelevel of antibiotics in the culture media can be increased withoutcausing death of the production cells, thereby increasing the efficiencyof industrial antibiotic production methods. Suitable polynucleotidesthat can be used to transform antibiotic-producing bacteria can containnucleic acid residues 34318-35184 of the polynucleotide having GenBankAccession No. gi2896719 or encode a polypeptide containing amino acidresidues 5717-4858 of Sanger Center Accession No. 684.

[0056] Suitable bacteria for use in the invention method for increasingproduction of antibiotics by antibiotic-producing bacteria includeStreptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsisorientalis, Streptomyces lavendulae, Streptomyces coelicolor,Streptomyces rochei and Saccharopolyspora erythraea.

[0057] In yet another embodiment according to the present invention,there are provided methods for detoxifying a toxic substance bycontacting the toxic substance with bacteria transformed with apolynucleotide that encodes an acyl glucosaminyl inositol amidase andexpressing the amidase in order to detoxify the toxic substance.Preferably, the bacteria is a strain currently in use for detoxificationof environmental pollutants and the bacteria are transformed with apolynucleotide that encodes the amidase such that the amidase isexpressed intracellularly under environmental conditions. Theenvironmental condition may include or be a pollutant. Suchenvironmental pollutants that may be detoxified according to inventionmethods include, but are not limited to 1, 2 dibromoethane, 1,2dichloroethane, perchloroethene, trichloroethene, isoprene, and vinylchloride. They are from pesticides, industrial degreasers, solvents, andfrom the production polyvinyl chloride polymers (plastics), such as ahalogenated hydrocarbon, or the epoxides, such as isoprene monoxide, andthe like.

[0058] In still another embodiment according to the present invention,there are provided processes for preparation of GlcN-Ins by contactingan N-acyl glucosaminyl inositol under suitable conditions with an acylglucosaminyl inositol amidase so as to hydrolyze the amide bond thereinto obtain stereochemically pure α(11) GlcN-Ins(1-D-myo-inosityl-2-amino-2-deoxy- α-D-glucopyranoside). Preferably theN-acyl glucosaminyl inositol is a mycothiol S-conjugate, such as thebimane derivative of mycothiol. The amidase cleaves the N-acylglucosaminyl inositol, freeing GlcN-Ins as one of the cleavage breakdownproducts. GlcN-Ins has utility in conducting research regarding amidaseactivity and mycothiol biochemistry in bacteria, development of productsand procedures for overcoming the antibiotic resistance of pathogenicbacteria, such as actinomycetes, and as a precursor for formation ofacyl glucosaminyl inositol derivatives and inhibitors of amidasesthereof.

[0059] A “conservative variation” in an amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule, and provided that thepolypeptide essentially retains its functional properties. Aconservative amino acid substitution, for example, substitutes one aminoacid for another of the same class (e.g., substitution of onehydrophobic amino acid, such as isoleucine, valine, leucine, ormethionine, for another, or substitution of one polar amino acid foranother, such as substitution of arginine for lysine, glutamic acid foraspartic acid or glutamine for asparagine). One or more amino acids canbe deleted, for example, from an amidase polypeptide, resulting inmodification of the structure of the polypeptide, without significantlyaltering its biological activity. For example, carboxyl-terminal aminoacids that are not required for amidase activity can be removed.

[0060] Alternatively, an antisense oligonucleotide can be designed tohybridize under in vivo conditions with a messenger RNA that encodes apolypeptide having an N-terminal amino acid sequence as set forth in SEQID NO:2, or contains an amino acid segment as set forth in SEQ ID NOs:3,4, or 5, or a conservative variation thereof.

[0061] The antisense oligonucleotide can comprises from about 10 toabout 60 nucleic acid residues, for example from 10 to about 50, or from10 to about 40, 30 or 20 nucleic acid residues. “Hybridization” refersto the process by which a nucleic acid strand joins with a complementarystrand through base pairing. Hybridization reactions can be sensitiveand selective so that a particular sequence of interest will join with acomplementary strand even in samples in which it is present at lowconcentrations. Suitable intracellular conditions for hybridization ofan antisense oligonucleotide to messenger RNA will be determined by theparticular bacterium used in the invention method. In general, the pH,temperature and salt concentration must be comparable to intracellularconditions in the test bacterium.

[0062] Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention. In general,expression vectors containing promoter sequences which facilitate theefficient transcription of the inserted eukaryotic genetic sequence areused in connection with the host. The expression vector typicallycontains an origin of replication, a promoter, and a terminator, as wellas specific genes which are capable of providing phenotypic selection ofthe transformed cells.

[0063] In addition to expression vectors known in the art such asbacterial, yeast and mammalian expression systems, baculovirus vectorsmay also be used. One advantage to expression of foreign genes in thisinvertebrate virus expression vector is that it is capable of expressionof high levels of recombinant proteins, which are antigenically andfunctionally similar to their natural counterparts. Baculovirus vectorsand the appropriate insect host cells used in conjunction with thevectors will be known to those skilled in the art.

[0064] The term “recombinant expression vector” refers to a plasmid,virus or other vehicle known in the art that has been manipulated byinsertion or incorporation of the invention acyl glucosaminyl inositolamidase genetic sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertedgenetic sequence of the host. The expression vector typically containsan origin of replication, a promoter, as well as specific genes whichallow phenotypic selection of the transformed cells. Vectors suitablefor use in the present invention include, but are not limited to theT7-based expression vector for expression in bacteria (Rosenberg, etal., Gene, 56:125, 1987), the pMSXND expression vector for expression inmammalian cells (Lee and Nathans, J. Biol. Chem., 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

[0065] The vector may include a phenotypically selectable marker toidentify host cells which contain the expression vector. Examples ofmarkers typically used in prokaryotic expression vectors includeantibiotic resistance genes for ampicillin (β-lactamases), tetracyclineand chloramphenicol (chloramphenicol acetyltransferase). Examples ofsuch markers typically used in mammalian expression vectors include thegene for adenosine deaminase (ADA), aminoglycoside phosphotransferase(neo, G418), dihydrofolate reductase (DHFR),hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), andxanthine guanine phosphoribosyltransferse (XGPRT, gpt).

[0066] The isolation and purification of host cell expressedpolypeptides of the invention may be by any conventional means such as,for example, preparative chromatographic separations and immunologicalseparations such as those involving the use of monoclonal or polyclonalantibody.

[0067] Transformation of the host cell with the recombinant DNA may becarried out by conventional techniques well known to those skilled inthe art. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth and subsequently treated by electroporation orthe CaCl₂ method using procedures well known in the art. Alternatively,MgCl₂ or RbCl could be used.

[0068] Where the host used is a eukaryote, various methods of DNAtransfer can be used. These include transfection of DNA by calciumphosphate-precipitates, conventional mechanical procedures such asmicroinjection, insertion of a plasmid encased in liposomes, or the useof virus vectors. Eukaryotic cells can also be cotransformed with DNAsequences encoding the polypeptides of the invention, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory,Gluzman ed., 1982). Examples of mammalian host cells include COS, BHK,293, and CHO cells.

[0069] Eukaryotic host cells may also include yeast. For example, DNAcan be expressed in yeast by inserting the DNA into appropriateexpression vectors and introducing the product into the host cells.Various shuttle vectors for the expression of foreign genes in yeasthave been reported (Heinemann, J. et al., Nature, 340:205, 1989; Rose,M. et al., Gene, 60:237, 1987).

[0070] The invention provides antibodies which are specifically reactivewith invention amidase polypeptides or fragments thereof.

[0071] Antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations are provided. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler, et al., Nature, 256:495, 1975; CurrentProtocols in Molecular Biology, Ausubel, et al., ed., 1989). Monoclonalantibodies specific for acyl glucosaminyl inositol amidase polypeptidecan be selected, for example, by screening for hybridoma culturesupernatants which react with acyl glucosaminyl inositol amidasepolypeptides, but do not react with other bacterial amidases.

[0072] Antibody which consists essentially of pooled monoclonalantibodies with different epitopic specificities, as well as distinctmonoclonal antibody preparations are provided. Monoclonal antibodies aremade from antigen containing fragments of the protein by methods wellknown in the art (Kohler, et al., Nature, 256:495, 1975; CurrentProtocols in Molecular Biology, Ausubel, et al., ed., 1989).

[0073] The term “antibody” as used in this invention includes intactmolecules as well as fragments thereof, such as Fab, F(ab′)₂ and Fvwhich are capable of binding the epitopic determinant. These antibodyfragments retain some ability to selectively bind with its antigen orreceptor and are defined as follows:

[0074] (1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

[0075] (2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

[0076] (3) (Fab′)2, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

[0077] (4) Fv, defined as a genetically engineered fragment containingthe variable region of the light chain and the variable region of theheavy chain expressed as two chains; and

[0078] (5) Single chain antibody (“SCA”), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

[0079] Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1988), incorporated herein by reference).

[0080] As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics.

[0081] Antibodies which bind to acyl glucosaminyl inositol amidasepolypeptide of the invention can be prepared using an intact polypeptideor fragments containing small peptides of interest as the immunizingantigen. The polypeptide or a peptide used to immunize an animal can bederived from translated cDNA or chemical synthesis which can beconjugated to a carrier protein, if desired. Such commonly used carrierswhich are chemically coupled to the peptide include keyhole limpethemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanustoxoid. The coupled peptide is then used to immunize the animal (e.g., amouse, a rat, or a rabbit).

[0082] If desired, polyclonal or monoclonal antibodies can be furtherpurified, for example, by binding to and elution from a matrix to whichthe polypeptide or a peptide to which the antibodies were raised isbound. Those of skill in the art will know of various techniques commonin the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies (See forexample, Coligan, et al., Unit 9, Current Protocols in Immunology, WileyInterscience, 1994, incorporated herein by reference).

[0083] It is also possible to use the anti-idiotype technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

[0084] In yet other preferred embodiments, the recombinant acylglucosaminyl inositol amidase polypeptide is a fusion protein furthercomprising a second polypeptide portion having an amino acid sequencefrom a protein unrelated the acyl glucosaminyl inositol amidase. Suchfusion proteins can be functional in a two-hybrid assay.

[0085] Another aspect of the present invention provides a substantiallypure nucleic acid having a nucleotide sequence which encodes an acylglucosaminyl inositol amidase polypeptide, or a fragment thereof, havingan amino acid sequence at least 60% homologous to one of SEQ ID NOs:2,3, 4 or 5. In a more preferred embodiment, the nucleic acid encodes aprotein having an amino acid sequence at least 80% homologous to SEQ IDNO:2, more preferably at least 90% homologous to SEQ ID NO:2, and mostpreferably at least 95% homologous to SEQ ID NO:2.

[0086] In another embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides encoding SEQ ID NO:3; more preferably to atleast 20 consecutive nucleotides encoding SEQ ID NO:3; more preferablyto at least 40 consecutive nucleotides encoding SEQ ID NO:3.

[0087] In a further embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides encoding SEQ ID NO:4; more preferably to atleast 20 consecutive nucleotides encoding SEQ ID NO:4; more preferablyto at least 40 consecutive nucleotides encoding SEQ ID NO:4.

[0088] In yet a further embodiment, the nucleic acid hybridizes understringent conditions to a nucleic acid probe corresponding to at least12 consecutive nucleotides encoding SEQ ID NO:5; more preferably to atleast 20 consecutive nucleotides encoding SEQ ID NO:5; more preferablyto at least 40 consecutive nucleotides encoding SEQ ID NO:5.

[0089] Furthermore, in certain embodiments, the acyl glucosaminylinositol amidase nucleic acid will comprise a transcriptional regulatorysequence, e.g. at least one of a transcriptional promoter ortranscriptional enhancer sequence, operably linked to the acylglucosaminyl inositol amidase-gene sequence so as to render therecombinant acyl glucosaminyl inositol amidase gene sequence suitablefor use as an expression vector.

[0090] The present invention also features transgenic non-humanorganisms, e.g. bacteria which either express a heterologous S-conjugateamidase gene, or in which expression of their own acyl glucosaminylinositol amidase gene expression is disrupted. Such a transgenicorganism can serve as an model for studying acyl glucosaminyl inositolamidase activity and for screening for compounds that inhibit acylglucosaminyl inositol amidase activity in bacteria.

[0091] The present invention also provides a probe/primer comprising asubstantially purified oligonucleotide, wherein the oligonucleotidecomprises a region of nucleotide sequence which hybridizes understringent conditions to at least 10 consecutive nucleotides of sense orantisense sequence encoding one of the amino acid sequences encompassedby SEQ ID NOs:2, 3, 4 or 5, or naturally occurring mutants thereof.

[0092] Yet another aspect of the invention pertains to a peptidomimeticwhich binds to an acyl glucosaminyl inositol amidase polypeptide andinhibits its binding to S-conjugate-containing amide substrate. Forexample, a preferred peptidomimetic is an analog of a peptide having thesequence of one of the SEQ ID NOs. 1, 2, 3, 4, or 5. Non-hydrolyzablepeptide analogs of such residues can be generated using, for example,benzodiazepine, azepine, substituted gama-lactam rings, keto-methylenepseudopeptides, beta-turn dipeptide cores, or beta-aminoalcohols.

[0093] Other features and advantages of the invention will be apparentfrom the detailed description herein, and from the claims. The practiceof the present invention will employ, unless otherwise indicated,conventional techniques of cell biology, cell culture, molecularbiology, transgenic biology, microbiology, recombinant DNA, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, for example, Molecular Cloning ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (ColdSpring Harbor Laboratory Press:1989); DNA Cloning, Volumes I and II (D.N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984);Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D.Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D.Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I.Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984);the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds.,1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154and 155 (Wu et al. eds.), Immunochemical Methods In Cell And MolecularBiology (Mayer and Walker, eds., Academic Press, London, 1987); HandbookOf Experimental Immunology, Volumes I-IV (D. M. Weir and C. C.Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0094] As used herein, the term “nucleic acid” refers to polynucleotidessuch as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleicacid (RNA). The term should also be understood to include, asequivalents, analogs of either RNA or DNA made from nucleotide analogs,and, as applicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides.

[0095] As used herein, the terms “gene”, “recombinant gene” and “geneconstruct” refer to a nucleic acid comprising an open reading frameencoding an invention acyl glucosaminyl inositol amidase, including bothexon and (optionally) intron sequences. The term “intron” refers to aDNA sequence present in a given acyl glucosaminyl inositol amidase genewhich is not translated into protein and is generally found betweenexons.

[0096] “Homology” refers to sequence similarity between two peptides orbetween two nucleic acid molecules. Homology can be determined bycomparing a position in each sequence which may be aligned for purposesof comparison. When a position in the compared sequence is occupied bythe same base or amino acid, then the molecules are homologous at thatposition. A degree of homology between sequences is a function of thenumber of matching or homologous positions shared by the sequences.

[0097] The term “transfection” or “transforming” and grammaticalequivalents thereof, refers to the introduction of a nucleic acid, e.g.,an expression vector, into a recipient cell by nucleic acid-mediatedgene transfer. “Transformation”, as used herein, refers to a process inwhich a cell's genotype is changed as a result of the cellular uptake ofexogenous DNA or RNA, and, for example, the transformed cell expresses arecombinant form of one of the invention family of acyl glucosaminylinositol amidases.

[0098] “Cells” or “cell cultures” or “recombinant host cells” or “hostcells” are often used interchangeably as will be clear from the context.These terms include the immediate subject cell which expresses thecell-cycle regulatory protein of the present invention, and, of course,the progeny thereof. It is understood that not all progeny are exactlyidentical to the parental cell, due to chance mutations or difference inenvironment. However, such altered progeny are included in these terms,so long as the progeny retain the characteristics relevant to thoseconferred on the originally transformed cell. In the present case, sucha characteristic might be the ability to produce a recombinant acylglucosaminyl inositol amidase polypeptide.

[0099] As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. The term “expression vector” includes plasmids, cosmids orphages capable of synthesizing the subject acyl glucosaminyl inositolamidase polypeptide encoded by the respective recombinant gene carriedby the vector. Preferred vectors are those capable of autonomousreplication and/expression of nucleic acids to which they are linked. Inthe present specification, “plasmid” and “vector” are usedinterchangeably as the plasmid is the most commonly used form of vector.Moreover, the invention is intended to include such other forms ofexpression vectors which serve equivalent functions and which becomeknown in the art subsequently hereto.

[0100] “Transcriptional regulatory sequence” is a generic term usedthroughout the specification to refer to DNA sequences, such asinitiation signals, enhancers, and promoters, as well as polyadenylationsites, which induce or control transcription of protein coding sequenceswith which they are operably linked. In preferred embodiments,transcription of a recombinant acyl glucosaminyl inositol amidase geneis under the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the recombinant gene can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of the regulatory protein.

[0101] As used herein, a “transgenic organism ” is any organism,preferably a bacteria in which one or more of the cells of the organismcontain heterologous nucleic acid introduced by way of humanintervention, such as by transgenic techniques well known in the art.The nucleic acid is introduced into the cell by way of deliberategenetic manipulation, such as by microinjection or by infection with arecombinant virus or a vector. The term genetic manipulation does notinclude classical cross-breeding, or in vitro fertilization, but ratheris directed to the introduction of a recombinant DNA molecule. Thismolecule may be integrated within a chromosome, or it may beextrachromosomally replicating DNA. In the typical transgenic organismsdescribed herein, the transgene causes cells to express a recombinantform of the subject acyl glucosaminyl inositol amidase polypeptides.

[0102] As used herein, the term “transgene” means a nucleic acidsequence (encoding, e.g., an acyl glucosaminyl inositol amidasepolypeptide), which is partly or entirely heterologous, i.e., foreign,to the transgenic organism or cell into which it is introduced, or, ishomologous to an endogenous gene of the transgenic organism or cell intowhich it is introduced, but which is designed to be inserted, or isinserted, into the organism's genome in such a way as to alter thegenome of the cell into which it is inserted (e.g., it is inserted at alocation which differs from that of the natural gene or its insertionresults in a knockout). A transgene can include one or moretranscriptional regulatory sequences and any other nucleic acid, such asintrons, that may be necessary for optimal expression of a selectednucleic acid.

[0103] The term “evolutionarily related to”, with respect to nucleicacid sequences encoding acyl glucosaminyl inositol amidase polypeptides,refers to nucleic acid sequences which have arisen naturally in anorganism, including naturally occurring mutants. The term also refers tonucleic acid sequences which, while derived from a naturally occurringacyl glucosaminyl inositol amidase polypeptide, have been altered bymutagenesis, as for example, combinatorial mutagenesis, yet still encodepolypeptides which have the amidase activity of an acyl glucosaminylinositol amidase polypeptide.

[0104] One aspect of the present invention pertains to an isolatednucleic acid comprising the nucleotide sequence encoding an acylglucosaminyl inositol amidase polypeptide, fragments thereof encodingpolypeptides having acyl glucosaminyl inositol amidase activity, and/orequivalents of such nucleic acids. The term nucleic acid as used hereinis intended to include such fragments and equivalents. The termequivalent is understood to include nucleotide sequences encodingfunctionally equivalent acyl glucosaminyl inositol amidase polypeptidesor functionally equivalent peptides having an activity of an acylglucosaminyl inositol amidase polypeptide such as described herein.Equivalent nucleotide sequences will include sequences that differ byone or more nucleotide substitutions, additions or deletions, such asallelic variants; and will also include sequences that differ from thenucleotide sequence encoding native acyl glucosaminyl inositol amidasesdue to the degeneracy of the genetic code. Equivalents will also includenucleotide sequences that hybridize under stringent conditions (i.e.,equivalent to about 20-27° C. below the melting temperature of the DNAduplex formed in about 1 M salt) to the nucleotide sequence of an acylglucosaminyl inositol amidase gene, such as that as set forth in nucleicacid residues 34318-35184 of GenBank Accession No. gi3256022 or thepolynucleotide encoding amino acids residues 5717-4858 of Sanger Centerplasmid GMS-684 (SEQ ID NO:1), particularly those segments encoding thepolypeptides shown in one of SEQ ID NOs. 2, 3, 4, or 5. In oneembodiment, equivalents will further include nucleic acid sequencesderived from and evolutionarily related to such nucleotide sequences

[0105] The term “isolated” or “purified” as also used herein withrespect to nucleic acids, such as DNA or RNA, refers to moleculesseparated from other DNAs, or RNAs, respectively, that are present inthe natural source of the macromolecule. For example, an isolatednucleic acid encoding one of the subject acyl glucosaminyl inositolamidase polypeptides preferably includes no more than 10 kilobases (kb)of nucleic acid sequence which naturally immediately flanks the acylglucosaminyl inositol amidase gene in genomic DNA, more preferably nomore than 5 kb of such naturally occurring flanking sequences, and mostpreferably less than 1.5 kb of such naturally occurring flankingsequence. The term isolated or purified as used herein also refers to anucleic acid or peptide that is substantially free of cellular materialor culture medium when produced by recombinant DNA techniques, orchemical precursors or other chemicals when chemically synthesized.Moreover, an “isolated nucleic acid” is meant to include nucleic acidfragments which are not naturally occurring as fragments and would notbe found in the natural state.

[0106] In yet another embodiment, the nucleic acid of the inventionencodes a peptide having an amino acid sequence as shown in GenBankAccession CAA17198 or amino acid residues 5717-4858 of Sanger CenterAccession No. GMS-684. Preferred nucleic acids encode a peptide having aS-conjugate amidase polypeptide activity and being at least 60%homologous, more preferably 70% homologous and most preferably 80%homologous with an amino acid sequence shown in GenBank AccessionCAA17198 (encoded by nucleic acid residues 34318-35184 of GenBankAccession No. gi3256022) or with amino acid residues 5717-4858 of SangerCenter Accession No. GMS-684. Nucleic acids which encode peptides havingan activity of a S-conjugate amidase polypeptide and having at leastabout 90%, more preferably at least about 95%, and most preferably atleast about 98-99% homology with such amino acid sequences are alsowithin the scope of the invention.

[0107] Another aspect of the invention provides a nucleic acid whichhybridizes under high or low stringency conditions to a nucleic acidwhich encodes an acyl glucosaminyl inositol amidase polypeptide havingall or a portion of an amino acid sequence shown in one of SEQ ID NOs.2, 3, 4, or 5. Appropriate stringency conditions which promote DNAhybridization, for example, 6.0×sodium chloride/sodium citrate (SSC) atabout 45° C., followed by a wash of 2.0×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Forexample, the salt concentration in the wash step can be selected from alow stringency of about 2.0×SSC at 50° C. to a high stringency of about0.2×SSC at 50° C. In addition, the temperature in the wash step can beincreased from low stringency conditions at room temperature, about 22°C., to high stringency conditions at about 65° C.

[0108] Isolated nucleic acids which differ from the nucleotide sequencesdisclosed herein due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject acyl glucosaminyl inositol amidase polypeptides will exist amongprokaryotic cells. One skilled in the art will appreciate that thesevariations in one or more nucleotides (up to about 3-4% of thenucleotides) of the nucleic acids encoding a particular member of theacyl glucosaminyl inositol amidase polypeptide family may exist amongindividuals of a given species due to natural allelic variation. Any andall such nucleotide variations and resulting amino acid polymorphismsare within the scope of this invention.

[0109] Fragments of the nucleic acid encoding a biologically activeportion of the subject acyl glucosaminyl inositol amidase polypeptidesare also within the scope of the invention. As used herein, a fragmentof the nucleic acid encoding an active portion of an acyl glucosaminylinositol amidase polypeptide refers to a nucleotide sequence havingfewer nucleotides than the nucleotide sequence encoding the full lengthamino acid sequence of, for example, the S-conjugate amidasepolypeptides represented in nucleic acid residues 34318-35184 of GenBankAccession No. gi3256022, and which encodes a peptide which retains atleast a portion of the biological activity of the full-length protein(i.e., a peptide capable of acyl glucosaminyl inositol amidase activity)as defined herein, or alternatively, which is functional as anantagonist of the amidase activity of the fill-length protein. Nucleicacid fragments within the scope of the invention include those capableof hybridizing under high or low stringency conditions with nucleicacids from other species, e.g. for use in screening protocols to detecthomologs of the subject acyl glucosaminyl inositol amidase polypeptides.Nucleic acids within the scope of the invention may also contain linkersequences, modified restriction endonuclease sites and other sequencesuseful for molecular cloning, expression or purification of suchrecombinant peptides.

[0110] As indicated by the examples set out below, a nucleic acidencoding a peptide having an activity of a S-conjugate amidasepolypeptide may be obtained from mRNA or genomic DNA present in any of anumber of antibiotic-producing or pathogenic bacteria, particularlyactinomycetes, in accordance with protocols described herein, as well asthose generally known to those skilled in the art. A cDNA encoding anacyl glucosaminyl inositol amidase polypeptide, for example, can beobtained by isolating total mRNA from a bacterial cell. Double strandedcDNAs can then be prepared from the total mRNA, and subsequentlyinserted into a suitable plasmid or bacteriophage vector using any oneof a number of known techniques. A gene encoding an acyl glucosaminylinositol amidase polypeptide can also be cloned using establishedpolymerase chain reaction techniques in accordance with the nucleotidesequence information provided by the invention.

[0111] Another aspect of the invention relates to the use of an“antisense” isolated nucleic acid. As used herein, an “antisense”inhibition of endogenous production of an acyl glucosaminyl inositolamidase molecule is carried out by administration or in situ generationof oligonucleotide probes or their derivatives which specificallyhybridize (e.g. bind) under intracellular conditions, with the cellularmRNA and/or genomic DNA encoding an acyl glucosaminyl inositol amidasepolypeptide so as to inhibit expression of that protein or a constituentthereof, e.g. by inhibiting transcription and/or translation. Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix. In general,“antisense” therapy refers to the range of techniques generally employedin the art, and includes any therapy which relies on specific binding tooligonucleotide sequences.

[0112] An antisense construct of the present invention can be delivered,for example, as an expression plasmid which, when transcribed in thetransformed cell, produces RNA which is complementary to at least aunique portion of the cellular mRNA which encodes an acyl glucosaminylinositol amidase polypeptide. Alternatively, the antisense construct isan oligonucleotide probe which is generated ex vivo and which, whenintroduced into the cell causes inhibition of expression by hybridizingwith the mRNA and/or genomic sequences encoding one of the subject acylglucosaminyl inositol amidase proteins. Such oligonucleotide probes arepreferably modified oligonucleotides which are resistant to endogenousnucleases, e.g. exonucleases and/or endonucleases, and is thereforestable in vivo. Exemplary nucleic acid molecules for use as antisenseoligonucleotides are phosphoramidate, phosphothioate andmethylphosphonate analogs of DNA (see also U.S. Pat. Nos. 5,176,996;5,264,564; and 5,256,775). Additionally, general approaches toconstructing oligomers useful in antisense techniques have beenreviewed, for example, by van der Krol et al. (1988) Biotechniques6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

[0113] In addition, the oligomers of the invention may be used asreagents to detect the presence or absence of the target DNA or RNAsequences to which they specifically bind. Such diagnostic tests aredescribed in further detail below.

[0114] This invention also provides expression vectors comprising anucleotide sequence encoding a member of the invention family of acylglucosaminyl inositol amidase polypeptides and operably linked to atleast one regulatory sequence. Operably linked is intended to mean thatthe nucleotide sequence is linked to a regulatory sequence in a mannerwhich allows expression of the nucleotide sequence. Regulatory sequencesare art-recognized and are selected to direct expression of the peptidehaving an activity of an acyl glucosaminyl inositol amidase polypeptide.Accordingly, the term regulatory sequence includes promoters, enhancersand other expression control elements. Exemplary regulatory sequencesare described in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990). For instance,any of a wide variety of expression control sequences-sequences thatcontrol the expression of a DNA sequence when operatively linked to itmay be used in these vectors to express DNA sequences encoding the acylglucosaminyl inositol amidase polypeptides of this invention. Suchuseful expression control sequences, include, for example, the early andlate promoters of SV40, adenovirus or cytomegalovirus immediate earlypromoter, the lac system, the trp system, the TAC or TRC system, T7promoter whose expression is directed by T7 RNA polymerase, the majoroperator and promoter regions of phage lambda, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast .alpha.-mating factors, the polyhedron promoterof the baculovirus system and other sequences known to control theexpression of genes of prokaryotic or eukaryotic cells or their viruses,and various combinations thereof. It should be understood that thedesign of the expression vector may depend on such factors as the choiceof the host cell to be transformed and/or the type of protein desired tobe expressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other proteins encoded by thevector, such as antibiotic markers, should also be considered.

[0115] As will be apparent, the subject gene constructs can be used tocause expression of the subject acyl glucosaminyl inositol amidasepolypeptides in cells propagated in culture, e.g. to produce proteins orpeptides, including fusion proteins or peptides, for purification. Inaddition, recombinant expression of the subject acyl glucosaminylinositol amidase polypeptides in cultured antibiotic-producing cells,for example during large-scale production of antibiotics byantibiotic-producing bacteria, can be useful for increasing theresistance of the production cells to the toxic effect upon themselvesof the antibiotics they produce. Thus, the level of antibiotics in theculture media can be increased without causing death of the productioncells, thereby increasing the efficiency of industrial antibioticproduction methods.

[0116] This invention also pertains to a host cell transfected with arecombinant acyl glucosaminyl inositol amidase gene in order to expressa polypeptide having an activity of an acyl glucosaminyl inositolamidase polypeptide. The host cell may be any prokaryotic or eukaryoticcell. For example, an acyl glucosaminyl inositol amidase polypeptide ofthe present invention may be expressed in bacterial cells such as E.coli, insect cells (baculovirus), yeast, or mammalian cells. Othersuitable host cells are known to those skilled in the art.

[0117] Another aspect of the present invention concerns recombinant acylglucosaminyl inositol amidase polypeptides which are encoded by geneswhich have the amidase activity of an acyl glucosaminyl inositol amidasepolypeptide, or which are naturally occurring mutants thereof. The term“recombinant protein” refers to a protein of the present invention whichis produced by recombinant DNA techniques, wherein generally DNAencoding the acyl glucosaminyl inositol amidase polypeptide is insertedinto a suitable expression vector which is in turn used to transform ahost cell to produce the heterologous protein. Moreover, the phrase“derived from”, with respect to a recombinant gene encoding therecombinant acyl glucosaminyl inositol amidase polypeptide, is meant toinclude within the meaning of “recombinant protein” those proteinshaving an amino acid sequence of a native acyl glucosaminyl inositolamidase polypeptide, or an amino acid sequence similar thereto which isgenerated by mutations including substitutions and deletions of anaturally occurring acyl glucosaminyl inositol amidase polypeptide of aorganism.

[0118] The present invention further pertains to methods of producingthe subject acyl glucosaminyl inositol amidase polypeptides. Forexample, a host cell transfected with expression vector encoding one ofthe subject acyl glucosaminyl inositol amidase polypeptide can becultured under appropriate conditions to allow expression of the peptideto occur. The peptide may be secreted and isolated from a mixture ofcells and medium containing the peptide. Alternatively, the peptide maybe retained cytoplasmically and the cells harvested, lysed and theprotein isolated. A cell culture includes host cells, media and otherbyproducts. Suitable media for cell culture are well known in the art.The peptide can be isolated from cell culture medium, host cells, orboth using techniques known in the art for purifying proteins includingion-exchange chromatography, gel filtration chromatography,ultrafiltration, electrophoresis, and immunoaffinity purification withantibodies specific for particular epitopes of the subject acylglucosaminyl inositol amidase polypeptides.

[0119] Thus, a nucleotide sequence derived from the cloning of an acylglucosaminyl inositol amidase polypeptide of the present invention,encoding all or a selected portion of the protein, can be used toproduce a recombinant form of the protein via microbial cellularprocesses.

[0120] The recombinant acyl glucosaminyl inositol amidase polypeptidecan be produced by ligating the cloned gene, or a portion thereof, intoa vector suitable for expression in bacterial cells. Expression vehiclesfor production of a recombinant acyl glucosaminyl inositol amidasepolypeptide include plasmids and other vectors. For instance, suitablevectors include plasmids of the types: pBR322-derived plasmids,pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids andpUC-derived plasmids for expression in prokaryotic cells, such as E.coli.

[0121] A number of vectors exist for the expression of recombinantproteins in yeast. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, andYRP17 are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein). These vectorscan replicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

[0122] The various methods employed in the preparation of the plasmidsand transformation of host organisms are well known in the art. Forother suitable expression systems for both prokaryotic and eukaryoticcells, as well as general recombinant procedures, see Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1989) Chapters 16 and 17. In someinstances, it may be desirable to express the recombinant acylglucosaminyl inositol amidase polypeptide by the use of a baculovirusexpression system. Examples of such baculovirus expression systemsinclude pVL-derived vectors (such as pVL 1392, pVL 1393 and pVL941),pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors(such as the .beta.-gal containing pBlueBac III).

[0123] This invention further contemplates a method of generating setsof combinatorial mutants of the present acyl glucosaminyl inositolamidase polypeptides, as well as truncation mutants, and is especiallyuseful for identifying potential variant sequences (e.g. homologs) thatare functional in cleaving S-conjugate amide molecules. In arepresentative embodiment of this method, the amino acid sequences for apopulation of acyl glucosaminyl inositol amidase polypeptide homologsare aligned, preferably to promote the highest homology possible. Such apopulation of variants can include, for example, homologs from one ormore species, or homologs from the same species but which differ due tomutation. Amino acids which appear at each position of the alignedsequences are selected to create a degenerate set of combinatorialsequences. The presence or absence of amino acids from an alignedsequence of a particular variant is relative to a chosen consensuslength of a reference sequence, which can be real or artificial. Inorder to maintain the highest homology in alignment of sequences,deletions in the sequence of a variant relative to the referencesequence can be represented by an amino acid space (*), whileinsertional mutations in the variant relative to the reference sequencecan be disregarded and left out of the sequence of the variant whenaligned.

[0124] Further expansion of the combinatorial library can be made by,for example, by including amino acids which would represent conservativemutations at one or more of the degenerate positions. Inclusion of suchconservative mutations can give rise to a library of potentialcell-cycle regulatory sequences wherein Xaa(1) represents Ser, Thr, Asnor Gln; Xaa(2) represents Gly, Ala, Val, Leu, or Ile; Xaa(3) representsArg, Lys or His; Xaa(4) represents Gly, Ala, Val, Leu, Ile, Asp or Glu;Xaa(5) represents Gly, Ala, Val, Leu, Ile, Asn or Gln; Xaa(6) representsArg, Lys, His, Tyr or Phe; Xaa(7) represents Asp or Glu; Xaa(8)represents Pro, Gly, Ser or Thr; Xaa(9) represents Gly, Ala, Val, Leu,Ile, Asp or Glu; Xaa(10) represents Gly, Ala, Val, Leu, Ile, or an aminoacid gap; Xaa(11) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;Xaa(12) represents Phe, Tyr, Trp or an amino acid gap; Xaa(13)represents Ser or Thr; Xaa(14) represents Gly, Ala, Val, Leu, Ile, Arg,Lys or His; Xaa(15) represents Gly, Ala, Val, Leu, Ile, Ser or Thr;Xaa(16) represents Gly, Ala, Val, Leu or Ile; Xaa(17) represents Glx;Xaa(18) represents Gly, Ala, Val, Leu, Ile, Lys, His or Arg; Xaa(19)represents Arg or Gln; Xaa(20) represents Gly, Ala, Val, Leu or Ile;Xaa(21) represents Gly, Ala, Val, Leu or Ile; Xaa(22) represents Gly,Ala, Val, Leu, Ile, Lys, His or Arg; Xaa(23) represents Gly, Ala, Val,Leu, Ile, Thr or Ser; Xaa(24) represents Gly, Ala, Val, Leu, Ile, Ser,Thr or an amino acid gap, where in this context, an amino acid gap isunderstood to mean the deletion of that amino acid position from thepolypeptide. Alternatively, amino acid replacement at degeneratepositions can be based on steric criteria, e.g. isosteric replacement,without regard for polarity or charge of amino acid sidechains.Similarly, completely random mutagenesis of one or more of the variantpositions (Xaa) can be carried out.

[0125] In a preferred embodiment, the combinatorial acyl glucosaminylinositol amidase library is produced by way of a degenerate library ofgenes encoding a library of polypeptides which each include at least aportion of potential acyl glucosaminyl inositol amidase polypeptidesequences. For instance, a mixture of synthetic oligonucleotides can beenzymatically ligated into gene sequences such that the degenerate setof potential acyl glucosaminyl inositol amidase nucleotide sequences areexpressible as individual polypeptides, or alternatively, as a set oflarger fusion proteins (e.g. for phage display) containing the set ofacyl glucosaminyl inositol amidase polypeptide sequences therein.

[0126] There are many ways by which the library of potential acylglucosaminyl inositol amidase homologs can be generated from adegenerate oligonucleotide sequence. Chemical synthesis of a degenerategene sequence can be carried out in an automatic DNA synthesizer, andthe synthetic genes then be ligated into an appropriate gene forexpression. The purpose of a degenerate set of genes is to provide, inone mixture, all of the sequences encoding the desired set of potentialacyl glucosaminyl inositol amidase sequences. The synthesis ofdegenerate oligonucleotides is well known in the art (see for example,Narang, S A (1983) Tetrahedron 39:3; Itakura et al. (1981) RecombinantDNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. A G Walton,Amsterdam: Elsevier pp. 273-289; Itakura et al. (1984) Annu. Rev.Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477.

[0127] A wide range of techniques are known in the art for screeninggene products of combinatorial libraries made by point mutations, and,for that matter, for screening cDNA libraries for gene products having acertain property. Such techniques will be generally adaptable for rapidscreening of the gene libraries generated by the combinatorialmutagenesis of acyl glucosaminyl inositol amidase homologs. The mostwidely used techniques for screening large gene libraries typicallycomprises cloning the gene library into replicable expression vectors,transforming appropriate cells with the resulting library of vectors,and expressing the combinatorial genes under conditions in whichdetection of a desired activity facilitates relatively easy isolation ofthe vector encoding the gene product was detected. Each of theillustrative assays described below are amenable to high through-putanalysis as necessary to screen large numbers of degenerate sequencescreated by combinatorial mutagenesis techniques.

[0128] The invention also provides for reduction of the subject acylglucosaminyl inositol amidase polypeptides to generate mimetics, e.g.peptide or non-peptide agents, which are able to mimic binding of theauthentic acyl glucosaminyl inositol amidase polypeptide to a substrateS-conjugate amide molecule. Such mutagenic techniques as describedabove, as well as the thioredoxin system, are also particularly usefulfor mapping the determinants of an acyl glucosaminyl inositol amidasepolypeptide which participate in protein-protein interactions involvedin, for example, binding of the subject acyl glucosaminyl inositolamidase polypeptide to a substrate. To illustrate, the critical residuesof a subject acyl glucosaminyl inositol amidase polypeptide which areinvolved in molecular recognition of substrate can be determined andused to generate acyl glucosaminyl inositol amidase-derivedpeptidomimetics which bind to S-conjugate amide substrates and, like theauthentic acyl glucosaminyl inositol amidase polypeptide, cleave thesubstrate molecule, for example by amide hydrolase activity. Byemploying, for example, scanning mutagenesis to map the amino acidresidues of a particular acyl glucosaminyl inositol amidase polypeptideinvolved in binding to a substrate, peptidomimetic compounds (e.g.diazepine or isoquinoline derivatives) can be generated which mimicthose residues in binding to the amidase. For instance, non-hydrolyzablepeptide analogs of such residues can be generated using benzodiazepine(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), azepine(e.g., see Huffman et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,1985), beta-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231), and.beta.-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419; and Dann et al. (1986) Biochem Biophys Res Commun 134:71).

[0129] In the studies described herein, it was discovered thatalkylation of mycothiol with mBBr produces the stable, fluorescentderivative MSmB which can be quantitated by HPLC (Newton, et al. (1996),Newton, et al. (1995) supra.). However, when a pure sample of MSmB wasexposed to a cell-free extract from M. smegmatis, the recovery of MSmBwas poor and a substantial amount of AcCySmB was detected, indicatingthat MSmB had been cleaved by an enzyme present in the cell extract. Theexperiment was repeated using a partially purified cell extract andsamples of the incubation mixture were analyzed at intervals for theother potential product of MSmB cleavage, GlcN-Ins, as well as for MSmBand AcCySmB. Based upon 4 determinations at >50% conversion it was foundthat 1.0 equivalent of MSmB (0.1 nmol) yielded 1.00±0.02 equivalent ofAcCySmB and 0.80±0.08 equivalent of GlcN-Ins with the reactionproceeding to 97% conversion of MSmB in 60 min at 23° C. Thisestablished the presence in the cell extract of mycothiol S-conjugateamidase which catalyzes the reaction shown in FIG. 1C.

[0130] Since mBBr is known to penetrate cells rapidly and to convertintracellular thiols to their bimane derivatives (Newton, et al. (1995)supra.), the fate of mycothiol in M. smegmatis cells treated with mBBrwas examined to ascertain whether the reaction shown in FIG. 1C occursin vivo. A logarithmic phase culture of M. smegmatis was cooled on iceto ˜3° C. to reduce enzymatic reactions prior to reaction with 0.5 mMmBBr. Excess reagent was reacted with 2-mercaptoethanol and the cellswere pelleted, washed, and resuspended in a small amount of ice coldmedium. The cells were diluted with prewarmed medium and replaced in theshaking incubator at 37° C. (time, t=0). Samples were removed andanalyzed for intracellular and extracellular mycothiol related compoundsover a 4 h interval (FIG. 3).

[0131] At t=0 no significant mycothiol or MSmB was found in cells ormedium but both contained significant levels of AcCySmB, indicating thatthe mycothiol had fully reacted with mBBr and hydrolyzed to thecorresponding AcCySmB derivative (FIG. 3), much of this conversionpresumably having occurred during the initial incubation on ice. At t=0,90 nmol per 100 mL (3.5 μmol per g RDW) of AcCySmB was present in thecells and 160 nmol per 100 mL was found in the medium. Within 5 min thecellular AcCySmB level had fallen to 8 nmol per 100 mL and the mediumlevel increased to 220 nmol per 100 mL. Subsequent analyses foundessentially all of the AcCySmB in the medium at a level of 225-230 nmolper 100 mL, accounting for 80-90% of the original cellular mycothiolcontent. No MSmB appears to be exported from cells as <1 nmol per 100 mL(<0.5%) was detected in the medium or the cell washes.

[0132] The cellular level of GlcN-Ins was comparable to that of AcCySmBat t=0 (FIG. 3) and represented a ˜25-fold increase above the normallevel of ˜0.1 μmol GlcN-Ins per g RDW (S. J. Anderberg, et al. (1998)supra.). The GlcN-Ins level declined slowly over the 4 h incubationwhile the mycothiol content increased from a nearly undetectable levelat t=0 to about half the normal cellular level after 4 h (FIG. 4). Theseresults indicate that GlcN-Ins produced by cleavage of MSmB is retainedby the cell and is utilized in the resynthesis of mycothiol. Nodetectable low molecular weight bimane derivatives remained in the cellsat 4 h. During the 4 h incubation the A600 value initially decreased˜15% but then recovered its initial value of 1.2. Continued incubationat 37 C resulted in a further increase to 1.6 at 8.5 h and a finalA₆₀₀˜2.6 at 30 h. Thus, at least one cell doubling occurred subsequentto the treatment with mBBr and the cells entered stationary phase at anormal cell density.

[0133] Although the mammalian glutathione-dependent and themycobacterial mycothiol-dependent systems produce the same finalproduct, the mammalian system is more complex. In mammals, intracellularconversion of alkylating agents to glutathione S-conjugates is catalyzedby glutathione S-transferases (B. Ketterer, et al. (1988) in GlutathioneConjugation: Mechanisms and Biological Significance (H. Sies, et al.Eds.) pp 73-135, London, B. Mannervik, et al. (1988) CRC Crit. Rev.Bioch. 23:283-337) and occurs in various tissues. GlutathioneS-conjugates are exported to the plasma and transported to othertissues, notably kidney and liver, where they are extracellularlydegraded by y-glutamyltranspeptidase to CySR-Gly and the latter cleavedby a dipeptidase to produce a cysteine S-conjugate, CySR (J. L. Stevens,et al. (1989) supra.). CySR is imported and acetylated by acetyl CoA toproduce a mercapturic acid (AcCySR) which is ultimately excreted inurine and bile.

[0134] Although the final excreted product is the same in the twosystems, the detailed biochemistry is different. Intracellulardegradation of MSmB is advantageous for a single cell organism becausethe hydrophilic GlcN-Ins produced is retained by the cell and can beutilized for resynthesis of mycothiol. The more hydrophobic mercapturicacid, AcCySmB, is rapidly lost from cell. This loss may occur by passivediffusion or could be facilitated by a specific export system. SinceAcCys is a component of mycothiol, the mycothiol-dependentdetoxification of electrophiles requires only a single enzyme, mycothiolS-conjugate amidase, to cleave the S-conjugate and produce themercapturic acid excreted by the cell.

[0135] Other glutathione-producing cells excrete the bimane derivativeof glutathione (GSmB) intact. E. coli (A. Kaluzna, et al. (1977)Biochem. Mol. Biol. Int. 43:161-171), yeast (Z. Li, et al. (1996) J.Biol. Chem. 271:6509-6517), plants (E. Martinola, et al. (1993) Nature364:247-249), and cultured mammalian cells (R. P. J. Oude Elferrink, etal. (1993) Hepatology 17:434-444, T. Ishikawa, et al. (1994) J. Biol.Chem. 269:29085-29093) all excrete GSmB produced within the cell into avacuole or to the extracellular space using an ATP requiring ABCtransporter. Thus, the intracellular degradation of MSmB in mycobacteriarepresents a marked departure from the pattern found in GSH-producingorganisms.

[0136] Significant amidase activity was observed for a variety of groupsattached to sulfur in the S-conjugate (FIG. 8). Low but measurableactivity was obtained with the two carbon acetamido moiety attached tosulfur (FIG. 8, MSA) from reaction with iodoacetamide whereas 4-foldgreater activity was measured with an N-ethylsuccinimidyl residue (MSME)produced by reaction with N-ethylmaleimide. However, substantiallylarger maleimide derivatives (MSMC and MSMPB) exhibited only modestlyincreased activity. It is evident that invention S-conjugate amidase canaccommodate rather large groups attached to the sulfur. Nevertheless,MSmB was the best of the substrates tested and modification of thebimane by attachment of a positively charged trimethylammonio group(MSqB) or a negatively charged p-sulfobenzoyloxy residue (MSsB) led to a6-7-fold loss of activity (FIG. 8). The only S-conjugate studied thatwould occur in nature is that derived from cerulenin (MSC), anantibiotic produced by the actinomycete Cephalosporum cerulens. (S.Omura, (1981) supra.)

[0137] This mycothiol conjugate exhibited 8% of the activity found withMSmB. The S-conjugate structure shown in FIG. 8 was drawn on theassumption that the reaction of cerulenin with mycothiol produces aproduct analogous to that demonstrated previously for its reaction withcysteine (H. Funabashi, et al. (1989) J. Biochem. (Tokyo) 105:751-755).Based upon the finding that MSC is a reasonable substrate for inventionS-conjugate amidase, it is believed that invention S-conjugate amidasefunctions as a component of a mycothiol-dependent detoxification systemin mycobacteria which can operate to inactivate bacterial antibiotics.In this regard, it may be significant that an M. smegmatis mutantblocked in mycothiol production, and thus lacking the amidase cofactor,was found to have 20-fold increased sensitivity to rifampin Newton, etal. (1999), supra.).

[0138] Invention S-conjugate amidase has little activity with mycothiolor mycothiol disulfide (FIG. 8), which is an essential specificity inorder to minimize a futile cycle involving amidase degradation ofmycothiol or mycothiol disulfide in combination with mycothiolbiosynthesis. Although mycothiol is not a substrate for the amidase, atmM levels it does inhibit amidase activity with MSmB as substrate. Thethiol biotinylating reagent 3-(N-maleimidopropionyl)biocytin (MPB) isutilized to capture mycothiol as the MSMPB conjugate in our currentimmunoassay protocols for determination of mycothiol (M. D. Unson, etal. (1999) J. Clin. Microbiol. 37:2153-2157). Since MSMPB is a substratefor invention S-conjugate amidase (Table 2, FIG. 8), it is importantthat the amidase be inactivated when assaying cells by use of proteindenaturing conditions for cell extraction as employed here and in theearlier study (M. D. Unson, et al. (1999) supra.).

[0139] The invention family of acyl glucosaminyl inositol amidases is animportant practical tool for studies of mycothiol biochemistry becauseit provides an efficient means for producing GlcN-Ins. GlcN-Ins isrequired as a substrate for the assay of ATP-dependent cysteine:GlcN-Insligase (S. J. Anderberg, et al. (1998) supra., C. Bornemann, et al.(1997) supra.), as a standard for HPLC calibration (S. J. Anderberg, etal. (1998) supra.), and as a precursor of synthetic analogs. Mycothiolis easily isolated from M. smegmatis and can be converted quantitativelyto MSmB in minutes. Subsequent treatment with purified amidase producesan easily separated mixture of AcCySmB and stereochemically pure α(11)GlcN-Ins. This method is much faster and cheaper than the low yieldisolation from Micromonospora echinospora (S. J. Anderberg, et al.(1998) supra.) or the multi-step chemical synthesis (C. Bornemann, etal. (1997) supra.) previously used to produce GlcN-Ins, which generatesisomers of the final compound, only one of which is active.

[0140] Additional studies have been conducted to illustrate the use ofmycothiol by mycobacteria for detoxification of such toxic compounds asvinyl chloride, 1,2 dibromoethane, and numerous other haloalkanes(Example 9). Assay of the thiol content of Rhodococcus sp. Strain AD45shows that mycothiol is the major thiol, with glutathione as a minorthiol at about 10% of the mycothiol level. Further analysis of themycothiol S-conjugate amidase activity with mycothiol-bimane derivativeas substrate discovered that amidase activity was twice as high as thatfound in Mycobacterium smegmatis (see Example 9, Table 3). The highmycothiol content relative to the glutathione content of this organismalong with the inability to saturate the glutathione S-transferase withthe substrate glutathione suggests that the enzyme used by mycobacteriato detoxify toxic environmental substances, such as vinyl chloride, 1,2dibromoethane, numerous other haloalkanes, and the like, is actually amycothiol S-transferase and not a glutathione S-transferase.

[0141] Mycothiol S-conjugate amidase is present in the test organism (atlevels higher than found in M. smegmatis) and is believed to be involvedin the detoxification of the epoxide, isoprene monoxide, formed duringthe detoxification of isoprene by Rhodococcus sp. AD45. An example of arelated compound is the antibiotic cerulenin, an epoxide that reactswith mycothiol and is a substrate of mycothiol conjugate amidase derivedfrom M. smegmatis, as discussed above.

[0142] Thus, in another embodiment, bacteria used (or specificallyengineered) to detoxify environmental toxins can be transformed with thesubject gene constructs to cause or increase expression of acylglucosaminyl inositol amidase in the bacteria, thereby increasing thecapacity of the bacteria to detoxify environmental toxins or expandingthe range of toxins against which the bacteria are effective.

[0143] The invention will be further described with reference to thefollowing examples; however, it is to be understood that the inventionis not limited to such examples.

EXAMPLE 1

[0144] In vitro Enzymatic assays. The enzymatic activity was routinelyassayed by quantitation of the bimane derivative of N-acetylcysteine(AcCySmB) produced from the bimane derivative of mycothiol (MSmB),prepared from purified mycothiol (Newton et al., (1995) supra.).Separation of the various modified thiols was performed by high-pressureliquid chromatography (HPLC). A sample (2-10 μL) of extract was mixedwith 40 μL of 30 μM MSmB in 3 mM 2-mercaptoethanol, 25 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) chloride pH7.5 and reacted 10-30 min at 30° C. before quenching the reaction with50 μL of 40 mM methanesulfonic acid on ice. The mixture was centrifugedfor 3 min at 14,000×g in a microcentrifuge at room temperature and thesupernatant analyzed by HPLC without dilution. A shortened version (45min) of HPLC method 1 of Fahey and Newton (R. C. Fahey, et al. (1987)Methods Enzymol. 143:85-96) was used for separation of MSmB and AcCySmB.The bimane derivative of mycothiol eluted at 23.5 min and AcCySmB elutedat 27 min.

[0145] A preparation of the mycothiol S-conjugate amidase (30-50%saturated ammonium sulfate fraction chromatographed on Sephadex G-75)was used to study the stoichiometry of the reaction. Reaction wasinitiated by mixing a sample (9 μL, 7 μg total protein) of thepreparation of the enzyme with 0.9 mL of 50 mM sodium phosphate, pH 7.5containing 100 μM MSmB.

[0146] For determination of bimane derivatives of thiols, a sample (70μL) of reaction mixture was removed, mixed with 4 μL 5 M methanesulfonicacid, and analyzed by HPLC without dilution.

[0147] For analysis of GlcN-Ins, a sample (2-8 μL) of the reactionmixture was mixed with enough 1M HEPES chloride pH 8 to bring the volumeto 10 μl and then with 5 μL acetonitrile and 5 μL of 10 mM AccQ-Fluorreagent (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, Waters). Themixtures were incubated for 1 min at room temperature followed by 10 minat 60° C., diluted with 60 μL of water, and quantified by HPLC aspreviously described (S. J. Anderberg, et al. (1998) supra.).

[0148] The specificity of the amidase for substrate was assessed bymeasuring the production of GlcN-Ins in most cases. A sample (5 μL) of 1mM substrate was mixed with 40 μL of 3 mM 2-mercaptoethanol, 25 mM HEPESchloride, pH 7.5. The reaction was initiated with 5 μL of purifiedamidase (50-fold diluted stock, 4.4 μg ml³¹ ¹). Triplicate samples werequenched at 0, 10, and 30 min by mixing each sample with 50 μL ofacetonitrile containing 5 mM NEM and incubating at 60 C for 10 min.After cooling on ice, the samples were clarified by centrifugation for15 min at 14000 g. A sample (15 μL) of the supernatant was modified withAccQ-Fluor for amine analysis in a total reaction volume of 125 μL aspreviously described (S. J. Anderberg, et al. (1998) supra.).

[0149] In the substrate specificity tests, activity with 100 μMmycothiol or with the monobromobimane derivative of the compounds shownin Table 2 (chemical structures shown in FIG. 8) was assayed. In thesetests,1-D-myo-inosityl-2-(L-cysteinyl)-amido-2-deoxy-α-D-glucopyranoside(CySmB-GlcN-Ins) or of2-(N-acetyl-L-cysteinyl)amido-2-deoxy-(α,β)-D-glucopyranoside(AcCySmB-GlcN) was at least 10³ lower than with 100 μM MSmB,demonstrating that invention S-conjugate amidase is highly specific forS-conjugates of mycothiol. Conjugates of mycothiol with the antibioticcerulenin, N-ethylmaleimide, 3-(N-maleimidopropionyl)-biocytin, and7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin also exhibitedsignificant activity as shown in Table 2 below: TABLE 2^(a) ActivityAmidase Substrates (nmole/min/mg) Relative Activity (%) Mycothiolconjugate with: control^(b)  13 ± 11 0.29 ± 0.25 mBBr (MSmB) 4480 ± 870(100) sBBr (MSsB) 740 ± 94 17 ± 2  qBBr (MSqB) 620 ± 44 14 ± 1 cerulenin (MSC) 370 ± 20 8.0 ± 0.5 iodoacetamide (MSA) 21 ± 2 0.47 ±0.04 NEM (MSME) 107 ± 9  2.1 ± 0.2 CPM (MSMPC) 183 ± 50 3.8 ± 1.1 MPB(MSMPB) 246 ± 12 5.2 ± 0.3 Other substrates Mycothiol (MSH)  4 ± 1 0.1Mycothiol disulfide (MSSM) 25 ± 1 0.6 CySmB-GlcN-Ins <4.5^(c) <0.1AcCySmB-GlcN <0.8^(d) <0.02

[0150] mBBr=monobromobimane; MPB=3-(N-maleimidopropionyl)biocytin;MSA=iodoacetamide S-conjugate of mycothiol; MSC=cerulenin S-conjugate ofmycothiol; MSH=mycothiol,1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-α-D-glucopyranoside;MSME=NEM S-conjugate of mycothiol; MSMC=CPM S-conjugate of mycothiol,MSMPB=MPB S-conjugate of mycothiol; MSsB=sBBr S-conjugate of mycothiol;MSqB=qBBr S-conjugate of mycothiol; NEM=N-ethylmaleimide;qBBr=monobromotrimethylammoniobimane; sBBr=p-sulfobenzoyloxybromobimane.cl EXAMPLE 2

[0151] Preparation of GlcN-Ins. Salt free, stereochemically pureGlcN-Ins was prepared from MSmB by enzymatic hydrolysis using partiallypurified amidase. A sample of MSmB purified by HPLC (4.8 moles) wasincubated in 2 mL of water without buffer at 23° C. with 15 μg ofpartially purified amidase. The reaction was monitored hourly for itscontent of MSmB and AcCySmB and the pH was adjusted to 7.5 with 1M NaOHas necessary. Additional aliquots of enzyme were added as necessary toachieve complete hydrolysis of the MSmB over a maximum interval of 12 h,after which the reaction mixture was acidified to a pH less than 3 withtrifluoroacetic acid (Fluka). A 1 mL SepPak, C18 cartridge (Waters) wasprepared by sequentially washing with 5 mL of methanol; 5 mL of 50%methanol, 0.1% trifluoroacetic acid in water; and 20 mL 0.1%trifluoroacetic acid in water. The acidified reaction mixture wasapplied and the column eluted with 0.1% trifluoroacetic acid. Fractions(1 ml) were collected and analyzed for their content of GlcN-Ins. Thefractions containing GlcN-Ins were pooled, lyophilized, and resuspendedin a small volume of water. Complete hydrolysis of MSmB is importantbecause AcCys-mB and protein, but not MSmB, are retained on the SepPakC18 cartridge under these conditions.

EXAMPLE 3

[0152] Analysis of M. smegmatis treated in vivo with mBBr in culture.Since mBBr is known to penetrate cells rapidly and to convertintracellular thiols to their bimane derivatives (Newton, et al. (1995)supra.), the fate of mycothiol in M. smegmatis cells treated with mBBrwas examined to ascertain whether the reaction of FIG. 1C occurs invivo.

[0153] A logarithmic phase culture of M smegmatis was cooled on ice to˜3° C. to reduce enzymatic reactions prior to reaction with 0.5 mM mBBr.Excess reagent was reacted with 2-mercaptoethanol and the cells werepelleted, washed, and resuspended in a small amount of ice cold medium.The cells were diluted with prewarmed medium and replaced in the shakingincubator at 37° C. (time, t=0). Samples were removed and analyzed forintracellular and extracellular mycothiol related compounds over a 4 hinterval (FIG. 3).

[0154] At t=0 no significant mycothiol or MSmB was found in cells ormedium but both contained significant levels of AcCySmB, indicating thatthe mycothiol had fully reacted with mBBr and hydrolyzed to thecorresponding AcCySmB derivative (FIG. 3), much of this conversionpresumably having occurred during the initial incubation on ice. At t=0,90 nmol per 100 mL (3.5 μmol per g RDW) of AcCySmB was present in thecells and 160 nmol per 100 mL was found in the medium. Within 5 min thecellular AcCySmB level had fallen to 8 nmol per 100 mL and the mediumlevel increased to 220 nmol per 100 mL. Subsequent analyses foundessentially all of the AcCySmB in the medium at a level of 225-230 μmolper 100 mL, accounting for 80-90% of the original cellular mycothiolcontent. No MSmB appears to be exported from cells as <1 nmol per 100 mL(<0.5%) was detected in the medium or the cell washes. A logarithmicphase culture (1 L) of M. smegmatis mc²155 (OD₆₀₀=1.2) in 7H9Middlebrook medium was cooled on ice to 3° C. The iced culture wasincubated with mBBr (0.5 mM from a 180 mM stock solution inacetonitrile) for 20 min; excess 2-mercaptoethanol (1.0 mM) was added,and the incubation continued on ice for an additional 10 min. The cellswere pelleted by centrifugation and washed twice with 200 mL of sterile,ice-cold 7H9 Middlebrook medium to remove excess bimane derivative of2-mercaptoethanol. The cells were resuspended in 50 mL of sterile icecold medium and the experiment was initiated (t=0) by dilution into 950mL of prewarmed (37° C.) 7H9 Middlebrook medium. The cell suspension wasshaken in an incubator (225 rpm, 37° C.) and a “t=0” sample (100 mL) wasremoved from the culture within one min. This sample was mixed with anequal weight of ice and stored on ice until the second sample was takensimilarly at 5 min, after which both were pelleted by centrifugation at5000 g and 4° C. Additional samples were obtained in this manner attimes up to 4 hours. An aliquot (0.5 mL) of supernatant was mixed with0.5 mL of acetonitrile and incubated at 60° C. for 10 min. Aftercentrifugation the supernatant was assayed by HPLC for bimane-labeledthiols in the medium. The pellet (always iced) was separated into 3roughly equal parts in 1.5 mL microcentrifuge tubes and extracted for 10min in 3 ways as follows.

[0155] The first sample was extracted using 1 mL of 60° C.acetonitrile-water for determination of cellular thiol-bimanederivatives. The second sample was extracted using 1 mL of 60° C.acetonitrile-water containing 2 mM mBBr and 20 mMtris(hydroxymethyl)aminomethane (Tris) pH 8.0 for determination of thesum of each cellular thiol and thiol-bimane derivative. The third samplewas extracted using 1 mL of 60° C. acetonitrile-water containing 5 mMN-ethylmaleimide (NEM) and 10 mM HEPES chloride pH 7.5. All tubes werecentrifuged at 14000 g in a microcentrifuge and the supernatants removedfor analysis.

[0156] For determination of GlcN-Ins, 0.1 mL was removed fromsupernatant of the third sample and 15 μL was assayed in a total assayvolume of 125 μL as previously described (S. J. Anderberg, et al. (1998)supra.). The remaining 0.9 mL of supernatant of the third sample wasderivatized with 2 mM mBBr and 20 mM Tris pH 8.0 to serve as the NEMcontrol for the analysis of sample 2. All assay pellets were dried in avacuum oven and weighed to obtain the residual dry weight (RDW) forcalculation of results.

[0157] The cellular level of GlcN-Ins was comparable to that of AcCySmBat t=0 (FIG. 3) and represented a ˜25-fold increase above the normallevel of ˜0.1 μmol GlcN-Ins per g RDW (S. J. Anderberg, et al. (1998)supra.). The GlcN-Ins level declined slowly over the 4 h incubationwhile the mycothiol content increased from a nearly undetectable levelat t=0 to about half the normal cellular level after 4 h (FIG. 3). Theseresults indicate that GlcN-Ins produced by cleavage of MSmB is retainedby the cell and is utilized in the resynthesis of mycothiol. Nodetectable low molecular weight bimane derivatives remained in the cellsat 4 h. During the 4 h incubation the A₆₀₀ value initially decreased˜15% but then recovered its initial value of 1.2. Continued incubationat 37 C resulted in a further increase to 1.6 at 8.5 h and a finalA₆₀₀˜2.6 at 30 h. Thus, at least one cell doubling occurred subsequentto the treatment with mBBr and the cells entered stationary phase at anormal cell density.

EXAMPLE 4

[0158] Purification of mycothiol S-conjugate amidase. M. smegmatis cellswere cultured as above to late log phase, collected by centrifugation at5000 g, and frozen at ˜70 C until used. Thawed cell paste (100 gm) wasmixed with 500 mL of 3 mM 2-mercaptoethanol, 25 mM HEPES chloride, pH7.5 (assay buffer) without protease inhibitors and disrupted bysonication on ice. The extract was centrifuged for 30 min at 1 5000 g at4 C and the supernatant was mixed with saturated ammonium sulfate to 20%saturation and incubated for 1 h on ice. After centrifugation at 15000gand 4° C. for 30 min, the pellet was discarded. The supernatant wasadjusted to 50% saturated ammonium sulfate, incubated on ice overnight,and centrifuged for 30 min 10000 g at 4 C. The pellet was resolubilizedin 60 mL of cold assay buffer and dialyzed against assay bufferovernight at 4 C.

[0159] The dialyzed sample was applied to a 1.4×27 cm column ofToyopearl DEAE 650C (TosoHaas) and the column was washed with ˜3 columnvolumes of assay buffer at 4 C. The column was developed with a lineargradient in assay buffer from 0 to 0.4 M NaCl and the amidase activityeluted at ˜0.2 M NaCl. The active fractions were pooled and saturatedammonium sulfate was added to 20% saturation. After 1 h on ice thesolution was clarified by centrifugation for 30 min at 10000 g and thepellet was discarded. The supernatant was applied to a 1.4×27 cm columnof Phenyl Sepharose 4B (Sigma) equilibrated with 20% saturated ammoniumsulfate in assay buffer at 4° C. The column was washed with 5 columnvolumes 20% saturated ammonium sulfate followed by 5 column volumes of10% saturated ammonium sulfate, both in assay buffer. The PhenylSepharose 4B column was eluted in assay buffer with a linear gradientfrom 10% to 0% saturated ammonium sulfate and the amidase activityeluted at ˜1-2% saturated ammonium sulfate. The active fractions werepooled and concentrated at 4 C using a Biomax-50 (Millipore)ultrafilter.

[0160] The concentrated activity pool was applied to a Sephadex G-100(Pharmacia) column (1.8×88 cm) equilibrated with assay buffer. Themajority of the activity eluted at an estimated M_(r) of 36 000. Themost active peak fractions were pooled and concentrated on CentriconC-30 (Amicon) ultrafilters at 4 C. Purified amidase was stored in assaybuffer containing 20% glycerol at −70° C. for at least 12 months withoutsignificant loss of activity.

[0161] Results of these experiments to purify the enzyme responsible forcleavage of MSmB from M. smegmatis are shown in Table 1 below. TABLE 1Purification of M. smegmatis mycothiol S-conjugate amidase. TotalSpecific Purification Protein Activity^(a) Activity^(a) YieldPurification Step (mg) (units) (units/mg) (%) Factor Crude Extract 8,70010.6 0.0012 100 1 20-50% 3,600 6.3 0.0018 59 1.5 ammonium sulfate pelletDEAE 650C 2,100 7.5 0.0036 71 3.0 Phenyl 76 9.4 0.123 89 103 SepharoseSephadex 0.35 1.16 3.3 11 2,800 G-100

[0162] As shown in FIG. 4, the third step in the three stepchromatography of the 20-50% saturated ammonium sulfate fraction, thethree center fractions eluted from the main peak of the G-100chromatography had specific activities of ˜3,000 nmol/min-mg proteinwith 30 μM MSmB as substrate and were pooled to provide a pure amidasepreparation. This sample produced a single band on SDS gelelectrophoresis. Only the peak fractions of activity from the gelfiltration step were selected and this was the principle factor inreducing the overall yield to 11% as shown in Table 1 above.

EXAMPLE 5

[0163] Characterization of mycothiol S-conjugate amidase. The level ofpurification and subunit molecular weight was estimated on 12% SDSpolyacrylamide slab gel electrophoresis (U. K. Laemmli, (1970) Nature227:680-685) calibrated with broad range standards (Bio-Rad). The nativemolecular weight was estimated based upon 3 preparative scalechromatographies on Sephadex G-100 used as the final step ofpurification and described above. The column was calibrated with dextranblue, phosphorylase B, bovine serum albumin, ovalbumin, carbonicanhydrase, trypsin inhibitor, and lysozyme, all from Sigma.

[0164] Based on these studies a subunit M_(r) for the amidase of 36000was estimated.

[0165] A native M_(r) of 36000 was determined for the main peak ofactivity on 3 independent preparative Sephadex G-100 chromatographicseparations. This indicates that invention S-conjugate amidase is activeas a monomer. A small and variable amount of activity eluted in the voidvolume (FIG. 3), prior to bovine serum albumin (M_(r)=68000). Thus, anaggregate larger than a dimer, or a larger protein with relatedcatalytic activity, may be present. The activity from the precedingPhenyl Sepharose step was concentrated using a 50000 cutoff filter andno activity was found in the filtrate, suggesting that at high proteinconcentration the enzyme may be aggregated.

[0166] A value of K_(m)=95±8 μM and a value of k_(cat)=8 s⁻¹ wasdetermined for invention S-conjugate amidase with MSMB as substrate(Table 1).

[0167] The amino-terminal sequence of the purified M. smegmatis amidasewas determined on an Applied Biosystems Procise Model 494 gas phaseprotein sequencer by the UCSD Department of Biology Protein SequencingFacility. Sequencing of the amino-terminal portion of purified amidaseproduced an amino-terminal sequence of (M)SELRLMAVHAHPDDESSKG (SEQ IDNO:2). The first amino acid was not uniquely defined and its assignmentwas uncertain until later verified as methionine. A BLAST search (SangerCentre) of the M. tuberculosis H37Rv genome database (S. T. Cole, et al.(1998) Nature 393:537-544) identified an open reading frame of unknownfunction, Rv1082, having an identical amino-terminal sequence and aM_(r) of 32700. When M tuberculosis Rv1082 gene was used to BLAST searchthe available databases, open reading frames with identicalamino-terminal sequences and very high overall homology were found ingenomes of M. tuberculosis CSU #93 (TIGR), M avium(TIGR), M. leprae(Sanger Centre) and M. bovis (Sanger Centre). An alignment of thesequences of five homologs of M. Smegmatis mycothiol S-conjugate amidaseis shown in FIG. 6.

EXAMPLE 6

[0168] Preparation of Mycothiol S-conjugates. Mycothiol S-conjugateswere prepared by reaction of excess electrophile with mycothiol followedby removal or reaction of excess electrophile. Stock solutions (100 mM)of electrophile were prepared in acetonitrile (NEM, iodoacetamide;Sigma) or in dimethylsulfoxide[7-diethylamino-3-(4′-maleimidylphenyl)-4-methylcoumarin (CPM, MolecularProbes), 3-(N-maleimidopropionyl)biocytin (MPB, Sigma)]. Reaction withthese electrophiles at 2 mM in 25 mM *HEPES chloride pH 7.5 wasinitiated by addition of mycothiol to 1 mM from a 32 mM stock solutionin H₂O (pH ˜3) and was allowed to proceed 15 min in the dark. Excessreagent was removed by extracting 3 times with an equal volume ofH₂O-saturated dichloromethane. A cerulenin (Sigma) stock solution (100mM in acetonitrile) was diluted 5-fold in 25 mM HEPES chloride pH 7.5,reacted 15 min with 1 mM mycothiol, and extracted 5 times withH₂O-saturated dichloromethane. Prior to extraction a 1 μL aliquot waswithdrawn for reaction with mBBr and analysis of residual mycothiol byHPLC. This showed that >99% of the mycothiol had reacted. Stocksolutions (50 mM) of p-sulfobenzoyloxybromobimane (sBBr) andmonobromotrimethylammoniobimane (qBBr) were prepared in 50% aqueousdimethylsulfoxide and diluted to 2 mM for reaction with 1 mM mycothiolin 25 mM HEPES chloride pH 7.5 for 15 min. Since these chargedelectrophiles cannot be extracted with dichloromethane, reaction wasterminated with excess (1.1 mM) 2-mercaptoethanol. In control studies itwas shown that 100 μM levels of these 2-mercaptoethanol derivatives donot inhibit the amidase promoted hydrolysis of MSmB and it was assumedthat their presence does not influence the reaction rate with otherbimane derivatives of mycothiol.

EXAMPLE 7

[0169] Cysteine:GlcN-Ins Ligase assays. The purified M. smegmatisamidase was assayed for ATP-dependent cysteine:GlcN-Ins ligase activityessentially as described by Anderberg et al (S. J. Anderberg, et al.(1998) supra.). As a positive control, a cell extract was prepared fromM. smegmatis logarithmic phase cells disrupted by sonication on ice in 3mM 2-mercaptoethanol in 50 mM HEPES chloride pH 7.5. The cell debris waspelleted by centrifugation at 14000 g for 3 min and the supernatant wasdialyzed against 3 mM 2-mercaptoethanol in 50 mM HEPES chloride pH 7.5overnight at 4° C.

[0170] The purified amidase or dialyzed M. smegmatis extract wasincubated in 100 μM Cysteine, 50 μM GlcN-Ins, 1 mM ATP, 5 mM MgCl₂ in 50mM HEPES chloride pH 7.5 at 30° C. and assayed for the time dependentformation of1-D-myo-inosityl-2-(L-cysteinyl)amido-2-deoxy-α-D-glucopyranoside(Cys-GlcN-Ins) by HPLC (S. J. Anderberg, et al. (1998) supra.). Thereaction was initiated with the addition of the purified amidase (0.044μg) or cell extract (50 μg protein) and was sampled at 0 and 60 min.

[0171] The purified amidase (0.044 μg) gave <0.33 nmol/min/mgCys-GlcN-Ins at a protein concentration where the amidase reaction ratefor 30 μM MSmB was ˜3000 nmol/min/mg. As a positive control, the ligasereaction was also assayed for a dialyzed crude extract from M. smegmatisand 0.36 nmol/min/mg protein Cys-GlcN-Ins was formed in accord withprevious reports (Newton, et al. (1999), supra., S. J. Anderberg, et al.(1998) supra.). Thus, mycothiol S-conjugate amidase does not appear tobe involved in mycothiol biosynthesis since it has no significantability to catalyze ATP-dependent ligation of cysteine with GlcN-Ins. Ittherefore does not appear to be a bifunctional enzyme analogous to theglutathionylspermidine synthetase/amidase which catalyzes both thebiosynthesis and degradation of glutathionylspermidine in E. coli (D. S.Kwon, et al.(1997) supra) and in Crithidia fasciculata (E. Tetaud, etal. (1998) supra.).

EXAMPLE 8 Cloning and Expression of M. tuberculosis Rv1082.

[0172] Bacterial strains, vectors and culture conditions. M.tuberculosis H37Rv NCTC 7416 was obtained from the National Collectionof Type Cultures, London, United Kingdom. E. coli DH5α (ClontechLaboratories, Inc., Palo Alto, Calif.) and E. coli BL21(DE3) (Novagen, R& D) were used for maintenance of plasmids and expression of foreignproteins, respectively. The plasmid pET-22b (Novagen) was used as anexpression vector in E. coli BL21 (DE3). E. coli strains were culturedon Luria-Bertani (LB) agar or broth with or without selectiveantibiotics. Mycobacterial strains were cultured in Middlebrook 7H9broth or 7H10 agar (Difco) supplemented with OADC (Difco) Tween 80, andglycerol.

[0173] Amplification and cloning of Rv1082. Genomic DNA of M.tuberculosis H37Rv was prepared as described previously (Newton, et al.(1999), supra.). The open reading frame Rv 1082 which encodes for theputative amidase was amplified from this DNA with the following primers:1, 5′-TAGCCATGGTGAGCGAACTGCGGTTGATG-3′ (SEQ ID:10); and 2,5′-GGATCCCGATCCCGGCGAACAATTCGGT-3′ (SEQ ID:11). Primers 1 and 2contained NcoI and BamHI restriction sites respectively. PCR wasperformed with Taq polymerase obtained from Gibco Brl, using 2 mM MgCl₂and 5% Dimethyl sulfoxide (DMSO). Annealing temperatures was 50° C. ThePCR products were separated on a 1% agarose gel. The appropriate PCRproduct was ligated into the vector pCR2.1 of the TA cloning kit(Invitrogen) and transformed into E. coli DH5α or INVF′α by standardchemical transformation procedure. Clones containing the vector wereselected on LB+ampicillin (100 μg/ml) plates and plasmid DNA wasdigested with restriction endonucleases NcoI and BamHI (Fermentas).Restriction enzyme-digested plasmids were isolated with a QIAquick gelextraction kit (Qiagen Ltd.). A corresponding digestion was also appliedto plasmid pET-22b and the two products were ligated together with T4DNA ligase to obtain the plasmid pYA1082E (FIG. 2).

[0174] Expression and purification of mycothiol S-conjugate amidase.Competent cells of E. coli BL21 (DE3) were prepared according by theCaCl₂ method (30) and were transformed by the heat shock method for 2min at 42° C. with 100 ng of pYA1082E (FIG. 2). The transformed E. coliwere then plated onto LB agar supplemented with ampicillin (100 μg/ml).Single colonies were inoculated into 5 ml of LB broth also containingampicillin (100 μg/ml). After over-night incubation at 37° C. withshaking, the individual cultures were diluted 1:50 in the same mediumand incubation was continued at 37° C. with shaking.Isopropyl-β-D-thiogalacto-pyranoside (IPTG) was added to a finalconcentration of 0.4 mM when the optical density (OD) at 600 nm reached0.6. Cultures were centrifuged at 5000×g, 15 minutes at roomtemperature, and pellets were sonicated three times for 30 seconds each.Proteins were separated by centrifugation (15,000×g, 4° C., 15 min) intosoluble and insoluble fractions.

[0175] Amidase inclusion bodies contained in the insoluble fractionswere purified from E. coli membrane proteins by centrifugation(45,000×g, 90 minutes, 4° C.). The invention amidase was separated by7.5% sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE)and stained with Coomassie blue. N-terminal amino acid sequence wasverified after electrophoresis of samples in SDS-PAGE andelectroblotting to PVDF membrane. Edman degradation was performed andthe sequence of the first five amino acids from the NH₂-terminus wasdetermined at the UBC Protein Sequencing Laboratory. In order to obtainsoluble protein, Rv 1082 amidase inclusion bodies were resuspended into1×PBS (pH 7.4) and slowly added drop-wise to solution of 16 M Urea and 2M DTT to make a final concentration of 8 M Urea and 1 M DTT. Solubleamidase was then dialyzed via a Spectra/P or 8000 cellulose membrane(VWR Scientific) against 200 volumes of 1×TBS (pH 7.4) at 4° C. for16-24 hours. The sample was then centrifuged for 15 minutes, 4°C.×15,000 g.

[0176] Cloning and expression of the M. tuberculosis Rv1082 gene. The M.tuberculosis Rv1082 gene was cloned and expressed under the control of aT7 promoter in the E. coli expression vector pET-22b. Using the primersdescribed in Materials and Methods, Rv1082 open reading frame wassuccessfully amplified by PCR from M. tuberculosis H37Rv genomic DNA togive a 858 bp fragment and cloned into the T7 expression vector pET-22b.The map of the resulting plasmid described as pYA1082E is shown in FIG.2. The mycothiol S-conjugate amidase gene of M. tuberculosis wasexpressed from pYA1082E following treatment of exponentially growingpYA1082E- E. coli BL21 (DE3) transformed cells with 1 mM IPTG at roomtemperature for about 12 hours . As shown by SDS-PAGE and Coomassie bluestaining, IPTG induced a protein approximately 37 kDa in size. Thisexpressed band was visible in both whole cell lysates andpost-sonication pellet within two hours of IPTG induction. Furtherpurification attempts revealed that mycothiol S-conjugate amidase waspresent in the form of insoluble inclusion bodies. The inclusion bodiesremained as stable insoluble aggregates even following multiple washeswith detergent solutions. To verify that the recombinant protein presentin the inclusion bodies is identical to the predicted protein encoded bythe M tuberculosis mycothiol S-conjugate amidase gene, N-terminal aminoacid sequencing on the IPTG-inducible protein we performed. The firstfive amino acids of this IPTG-induced band were shown to be identical tothe amino acid sequence of the Rv1082 gene product derived from the M.tuberculosis genome sequence database.

[0177] Purification and renaturation of mycothiol S-conjugate amidasefrom inclusion bodies. The approach taken involved solublization of theinclusion bodies in a highly concentrated urea and DTT solution followedby these values are similar to those obtained for other recombinantproteins that were recovered from inclusion bodies formed in E. coli(Landman et al. 1991). Cells were cultured in Luria Bertani brothcontaining 100 μg/mL ampicillin at 37° C. to OD₆₀₀=0.8 whenisopropyl-β-D-thiogalactopyranoside added to 0.4 mM and the culture wasshaken overnight at 25° C. Cells were pelleted by centrifugation for 10min at 5000 g and 4° C., sonicated in 5 volumes of assay buffer on ice,and centrifuged for 5 min at 14000 g at room temperature. About 85% ofthe amidase activity, using MSmB as substrate, was associated with thepellet fraction which was resuspended and incubated with periodicvortexing for 1 h at 37° C. in 8 M urea containing 20 mM DTT. Thesuspension was centrifuged for 3 min at 14000 g and the supernatantdialyzed against 100 volumes of 25 mM HEPES chloride pH 7.5 containing 1mM glutathione disulfide and 2 mM glutathione for 15 h, and then against100 volumes of 25 mM HEPES chloride pH 7.5 containing 3 mM2-mercaptoethanol for 4 h. After centrifugation, the supernatantcontained soluble amidase activity and was assayed with 0.1 mM mycothioland 0.1 mM MSmB as described above.

[0178]M. tuberculosis mycothiol S-conjugate amidase possesses functionalmycothiol-S-conjugate amide hydrolase activity. Alkylation of MSH withmBBr produces the fluorescent S-conjugate, MSmB, which can bequantitated by HPLC with fluorescence detection (Newton et al. 1995;Newton et al. 1996). E coli has no mycothiol metabolism and is notexpected to contain mycothiol conjugate amidase endogenous proteins thatwould give background to these assays. The amidase activity was found tobe associated with the insoluble cell pellet material. Using 0.1 mM MSmBas substrate, the resolublilzed crude protein extract was found toproduce 4.1±0.05 nmoles/min/mg protein GlcN-Ins and 5.4±0.3nmoles/min/mg protein AcCysmB.

[0179] Functional and Comparative analysis of mycothiol S-conjugateamidase. To verify the assignment of M. tuberculosis Rv1082 as amycothiol conjugate amidase, extracts from uninduced cells showed <0.01nmol/min/mg amidase activity with 100 μM MSMB as substrate whereasextracts of cells induced with isopropyl-β-D-thiogalactopyranosideproduced 4.1 nmol/min/mg amidase activity. E. coli, unlike M. smegmatis,does not have mycothiol metabolism (Newton, et al., 1996), whichdifference accounts for the very low background activity of uninducedcell extract. When these extracts were assayed with 0.1 mM mycothiol assubstrate, <0.002 nmol/min/mg amidase activity was observed. Thus, therecombinant M. tuberculosis amidase is more than 2000 fold more activewith MSmB than with mycothiol itself, a substrate specificity verysimilar to that of M. smegmatis amidase.(Table 2).

[0180] The S-conjugate amidase which is encoded by the M. tuberculosisopen reading frame Rv1082 is 288 amino acid long, slightly negativelycharged peptide with a predicted molecular weight of 32699 da andtheoretical PI of 5.11.

EXAMPLE 9

[0181] In this example, a thiol analysis of Rhodococcus sp. Strain AD45was conducted. The thiol analysis shows that mycothiol is the majorthiol, with glutathione as a minor thiol at about 10% of the mycothiollevel. Further analysis of the mycothiol S-conjugate amidase from thisbacterium showed amidase activity with mycothiol-bimane derivative assubstrate was 2-fold higher than that found in Mycobacterium smegmatis(Table 3). The high mycothiol content relative to the glutathionecontent of this organism along with the inability to saturate theglutathione S-transferase with the substrate glutathione suggests thatthe active amidase in this organism is actually a mycothiolS-transferase and not a glutathione S-transferase. Thus, the mycothiolS-conjugate amidase present in this organism (at levels higher thanfound in M. smegmatis) (Table 3) is believed to be involved in thedetoxification of the epoxide, isoprene monoxide, formed during thedetoxification of isoprene by Rhodococcus sp. AD45. An example of arelated compound is the antibiotic cerulenin, an epoxide that reactswith mycothiol and is a substrate of mycothiol conjugate amidase from M.smegmatis. Table 3 below shows the thiol content and mycothiolS-conjugate amidase in crude cell extracts of Rhodococcus sp. AD45 andMycobacterium smegmatis mc²155. TABLE 3 Amidase Thiol ContentμMoles/gram activity residual dry weight Units/mg Organism CysteineGlutathione Mycothiol protein Rhodococcus sp. 0.31 1.1 12.6 0.0025 AD45M. smegmatis 0.16 <0.001 10.6 0.0012 mc²155^(a)

[0182] These findings illustrate that, in the case of mycobacteria,mycothiol is the major low molecular weight thiol and will form amycothiol conjugate. The product of this conjugation may still be toxicand is a substrate for the mycothiol conjugate amidase. Reaction of themycothiol conjugate with a mycothiol conjugate amidase enables theexcretion of the detoxified conjugate as a mercapturic acid. Therefore,mycothiol and mycothiol S-conjugate amidase are involved in and can beused for detoxification of halogenated hydrocarbons and otherenvironmental toxins.

[0183] Accordingly, in another embodiment according to the presentinvention, there are provided methods for detoxifying a toxic substanceby contacting the toxic substance with an acyl glucosaminyl inositolamidase. In a preferred embodiment, bacteria transformed with apolynucleotide encoding an invention amidase polypeptide, or a variantthereof, is used to express the amidase in situ under environmentalconditions and the toxic substance is an environmental pollutant, suchas a halogenated hydrocarbon, hydrocarbon, or other petroleumderivative.

[0184] While the invention has been described in detail with referenceto certain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1 15 1 288 PRT Mycobacterium smegmatis 1 Met Ser Glu Leu Arg Leu Met AlaVal His Ala His Pro Asp Asp Glu 1 5 10 15 Ser Ser Lys Gly Ala Ala ThrThr Ala Arg Tyr Ala Ala Glu Gly Ala 20 25 30 Arg Val Met Val Val Thr LeuThr Gly Gly Glu Arg Gly Asp Ile Leu 35 40 45 Asn Pro Ala Met Asp Leu ProGlu Val His Gly Arg Ile Ala Glu Val 50 55 60 Arg Arg Asp Glu Met Ala LysAla Ala Glu Ile Leu Gly Val Glu His 65 70 75 80 His Trp Leu Gly Phe ValAsp Ser Gly Leu Pro Glu Gly Asp Pro Leu 85 90 95 Pro Pro Leu Pro Asp GlyCys Phe Ala Leu Val Pro Leu Glu Glu Pro 100 105 110 Val Lys Arg Leu ValArg Val Ile Arg Glu Phe Arg Pro His Val Met 115 120 125 Thr Thr Tyr AspGlu Asn Gly Gly Tyr Pro His Pro Asp His Ile Arg 130 135 140 Cys His GlnVal Ser Val Ala Ala Tyr Glu Ala Ala Ala Asp His Leu 145 150 155 160 LeuTyr Pro Asp Ala Gly Glu Pro Trp Ala Val Gln Lys Leu Tyr Tyr 165 170 175Asn His Gly Phe Leu Arg Gln Arg Met Gln Leu Leu Gln Glu Glu Phe 180 185190 Ala Lys Asn Gly Gln Glu Gly Pro Phe Ala Lys Trp Leu Glu His Trp 195200 205 Asp Pro Asp Asn Asp Val Phe Ala Asn Arg Val Thr Thr Arg Val His210 215 220 Cys Ala Glu Tyr Phe His Gln Arg Asp Asp Ala Leu Arg Ala HisAla 225 230 235 240 Thr Gln Ile Asp Pro Lys Gly Asp Phe Phe His Ala ProIle Glu Trp 245 250 255 Gln Gln Arg Leu Trp Pro Thr Glu Glu Phe Glu LeuAla Arg Ala Arg 260 265 270 Val Pro Val Thr Leu Pro Glu Asp Asp Leu PheLys Gly Val Glu Pro 275 280 285 2 20 PRT Artificial sequence acylglucosaminyl inositol amidases N-terminal region with 80% homology 2 MetSer Glu Leu Arg Leu Met Ala Val His Ala His Pro Asp Asp Glu 1 5 10 15Ser Ser Lys Gly 20 3 7 PRT Artificial sequence domain 1 of an amidehydrolase 3 Xaa His Ala His Pro Asp Asp 1 5 4 5 PRT Artificial sequencedomain 3 of an amide hydrolase 4 Xaa Pro Asp His Xaa 1 5 5 8 PRTArtificial sequence domain 4 of an amide hydrolase 5 Ala Leu Xaa Xaa HisXaa Xaa Gln 1 5 6 862 DNA Mycobacterium smegmatis 6 ggctcgacccccttgaacag gtcgtcctcg ggcagcgtga ccggcacgcg ggcccgcgcg 60 agctcgaactcctcggtcgg ccacaaccgc tgctgccact cgatcggggc gtggaagaag 120 tcgcccttgggatcgatctg tgtcgcgtgc gcacgcaacg cgtcgtcacg ctggtggaag 180 tactccgcgcagtgcacgcg ggtggtcacc cggttggcga acacgtcgtt gtcgggatcc 240 cagtgctcgagccatttggc gaacgggccc tcctgcccgt tcttggcgaa ctcctcctgc 300 aggagctgcatgcgctgacg gaggaagcca tggttgtagt acagcttctg caccgcccac 360 ggctcaccggcgtcgggata cagcaggtgg tcggccgcgg cctcgtacgc ggccaccgac 420 acctggtggcagcggatgtg gtcgggatgc gggtaaccac cgttctcgtc gtatgtggtc 480 atcacgtgcgggcggaactc gcggatcacc cgcaccagac gcttgacggg ctcctcgagc 540 gggaccagggcgaaacaccc gtcgggcagc ggcggcagcg ggtcaccctc cggcaatccg 600 gagtcgacgaaacccagcca gtggtgctcg acacccagga tctcggccgc tttggccatc 660 tcgtcacggcgcacctcggc gatccggccg tggacctcgg gcaggtccat cgccggattg 720 agaatgtctccgcgctcgcc gccggtcagg gtcaccacca tgacgcgggc accctcggcc 780 gcgtagcgcgcggtggttgc cgcacccttg ctggactcgt cgtccgggtg ggcatgcacc 840 gccatcaaccgcagttcact ca 862 7 288 PRT Mycobacterium tuberculosis 7 Met Ser Glu LeuArg Leu Met Ala Val His Ala His Pro Asp Asp Glu 1 5 10 15 Ser Ser LysGly Ala Ala Thr Leu Ala Arg Tyr Ala Asp Glu Gly His 20 25 30 Arg Val LeuVal Val Thr Leu Thr Gly Gly Glu Arg Gly Glu Ile Leu 35 40 45 Asn Pro AlaMet Asp Leu Pro Asp Val His Gly Arg Ile Ala Glu Ile 50 55 60 Arg Arg AspGlu Met Thr Lys Ala Ala Glu Ile Leu Gly Val Glu His 65 70 75 80 Thr TrpLeu Gly Phe Val Asp Ser Gly Leu Pro Lys Gly Asp Leu Pro 85 90 95 Pro ProLeu Pro Asp Asp Cys Phe Ala Arg Val Pro Leu Glu Val Ser 100 105 110 ThrGlu Ala Leu Val Arg Val Val Arg Glu Phe Arg Pro His Val Met 115 120 125Thr Thr Tyr Asp Glu Asn Gly Gly Tyr Pro His Pro Asp His Ile Arg 130 135140 Cys His Gln Val Ser Val Ala Ala Tyr Glu Ala Ala Gly Asp Phe Cys 145150 155 160 Arg Phe Pro Asp Ala Gly Glu Pro Trp Thr Val Ser Lys Leu TyrTyr 165 170 175 Val His Gly Phe Leu Arg Glu Arg Met Gln Met Leu Gln AspGlu Phe 180 185 190 Ala Arg His Gly Gln Arg Gly Pro Phe Glu Gln Trp LeuAla Tyr Trp 195 200 205 Asp Pro Asp His Asp Phe Leu Thr Ser Arg Val ThrThr Arg Val Glu 210 215 220 Cys Ser Lys Tyr Phe Ser Gln Arg Asp Asp AlaLeu Arg Ala His Ala 225 230 235 240 Thr Gln Ile Asp Pro Asn Ala Glu PhePhe Ala Ala Pro Leu Ala Trp 245 250 255 Gln Glu Arg Leu Trp Pro Thr GluGlu Phe Glu Leu Ala Arg Ser Arg 260 265 270 Ile Pro Ala Arg Pro Pro GluThr Glu Leu Phe Ala Gly Ile Glu Pro 275 280 285 8 225 PRT Artificialsequence consensus sequence between Mycobacterium tuberculosis andMycobacterium smegmatis 8 Met Ser Glu Leu Arg Leu Met Ala Val His AlaHis Pro Asp Asp Glu 1 5 10 15 Ser Ser Lys Gly Ala Ala Thr Ala Arg TyrAla Glu Gly Arg Val Val 20 25 30 Val Thr Leu Thr Gly Gly Glu Arg Gly IleLeu Asn Pro Ala Met Asp 35 40 45 Leu Pro Val His Gly Arg Ile Ala Glu ArgArg Asp Glu Met Lys Ala 50 55 60 Ala Glu Ile Leu Gly Val Glu His Trp LeuGly Phe Val Asp Ser Gly 65 70 75 80 Leu Pro Gly Asp Pro Pro Leu Pro AspCys Phe Ala Val Pro Leu Glu 85 90 95 Leu Val Arg Val Arg Glu Phe Arg ProHis Val Met Thr Thr Tyr Asp 100 105 110 Glu Asn Gly Gly Tyr Pro His ProAsp His Ile Arg Cys His Gln Val 115 120 125 Ser Val Ala Ala Tyr Glu AlaAla Asp Pro Asp Ala Gly Glu Pro Trp 130 135 140 Val Lys Leu Tyr Tyr HisGly Phe Leu Arg Arg Met Gln Leu Gln Glu 145 150 155 160 Phe Ala Gly GlnGly Pro Phe Trp Leu Trp Asp Pro Asp Asp Arg Val 165 170 175 Thr Thr ArgVal Cys Tyr Phe Gln Arg Asp Asp Ala Leu Arg Ala His 180 185 190 Ala ThrGln Ile Asp Pro Phe Phe Ala Pro Trp Gln Arg Leu Trp Pro 195 200 205 ThrGlu Glu Phe Glu Leu Ala Arg Arg Pro Pro Glu Leu Phe Gly Glu 210 215 220Pro 225 9 862 DNA Mycobacterium tuberculosis 9 ggctcgatcc cggcgaacaattcggtctcc ggtgggcgcg cggggatacg cgagcgagcc 60 aactcgaatt cctcggtcggccacagccgc tcctgccagg caagcggggc ggcgaagaat 120 tcggcgttcg ggtcgatctgggtggcatgc gcgcgcaacg catcgtcgcg ttggctgaag 180 tatttcgagc actcgacccgggtggtcact cggctggtga gaaagtcatg gtcggggtcc 240 cagtacgcca gccattgttcgaatgggccg cgttggccgt gccgggcgaa ctcatcctgc 300 aacatctgca tccgctcccgcaggaagccg tggacgtagt acagcttgga caccgtccac 360 ggctcacccg cgtcgggaaaccggcaaaag tcaccggccg cctcgtaggc agccaccgaa 420 acctgatggc agcgaatgtggtcgggatgt gggtagccgc cgttctcgtc gtaggtggtc 480 atcacgtgcg gccgaaactcgcgaaccacc cgcaccagcg cctcggtgga cacctccagc 540 ggtacccgcg cgaagcagtcatcaggcagc ggtggcggta aatcaccctt aggtagcccg 600 gagtcgacga agcccagccaggtgtgctcg acaccgagga tctcggccgc cttggtcatc 660 tcgtcacgcc ggatctcggcgatgcgccca tgcacgtccg gcaggtccat cgccgggttg 720 aggatctcgc cgcgctcaccaccggtcaac gtcaccacca gcacgcgatg accctcgtcg 780 gcgtagcgcg ccagggtggccgcgcccttg ctggactcgt catcggggtg ggcgtgcacc 840 gccatcaacc gcagttcgct ca862 10 29 DNA Artificial sequence PRIMER for RV 1082 10 tagccatggtgagcgaactg cggttgatg 29 11 28 DNA Artificial sequence PRIMER for RV 108211 ggatcccgat cccggcgaac aattcggt 28 12 24 PRT Artificial sequenceCOMBINATORIAL LIBRARY 12 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 13 270 PRTStreptomyces lincolnensis 13 Met Thr Gln Cys Leu Leu Thr Val His Ala HisPro Asp Asp Glu Ala 1 5 10 15 Ser Arg Gly Gly Ala Thr Val Ala His TyrThr Ala Gln Gly Val Arg 20 25 30 Ala Val Leu Val Thr Cys Thr Asp Gly GlyAla Gly Glu Val Leu Asn 35 40 45 Pro Ala Val Thr Asp Asp Phe Thr Pro GluArg Phe Val Ala Val Arg 50 55 60 Ser Ala Glu Leu Asp Ala Ser Ala Arg AsnLeu Gly Tyr Ser Ala Val 65 70 75 80 His Arg Leu Gly Tyr Arg Asp Ser GlyMet Asp Gly Thr Ala Gly Gly 85 90 95 Ala Glu Ala Phe Val Arg Ala Pro LeuAsp Glu Ala Ala Thr Arg Leu 100 105 110 Ala Arg Val Ile Ala Asp Glu ArgPro Asp Val Val Ile Gly Tyr Gly 115 120 125 Thr Asn His Thr Arg Asp ProHis Pro Asp His Ile Arg Ala Asn Glu 130 135 140 Val Leu Thr Arg Arg ValAsp Leu Leu Asp His Thr Pro Ala Val Tyr 145 150 155 160 His Ile Ala PheSer Arg Arg Arg His Arg Ala Leu His Gln Ala Cys 165 170 175 Val Asp SerGly Val Pro Ser Pro Tyr Glu Gly Gly Leu Ser Ala Pro 180 185 190 Pro GlyAla Phe Asp Asp Glu Trp Ile Thr Thr Leu Val Asp Val Thr 195 200 205 LysGly Asp Ala Val Glu Arg Arg Leu Asp Ala Leu Arg Ser His Val 210 215 220Thr Gln Val Pro Pro Ala Ser Gly Trp Phe Ala Leu Ser Pro Gln Gln 225 230235 240 Leu Arg Asp Ala Phe Pro Tyr Glu Glu Tyr Thr Arg Val Gly Ala Ala245 250 255 Pro Arg Glu Ala Val Val His Asp Leu Phe Thr Ala Pro Ala 260265 270 14 255 PRT Amycolatopsis mediterranei 14 Met Gly Thr Leu Val SerPhe His Ala His Pro Asn Asp Asp Thr Thr 1 5 10 15 Thr Cys Gly Gly ValLeu Arg Lys Ala His Glu Asp Gly His Arg Val 20 25 30 Val Leu Val Leu AlaThr Arg Gly Glu Leu Gly Tyr Asn Pro Asp Gly 35 40 45 Leu Leu Ala Glu GlyGlu Thr Leu Gly Asp Arg Arg Ala Val Glu Ala 50 55 60 Arg Ala Ala Ala AspVal Leu Gly Val Asp Arg Leu Glu Phe Leu Gly 65 70 75 80 Tyr Thr Asp SerGly Met Thr Ala Ala Ala Asp Gly Ala Gly Thr Phe 85 90 95 Gln Thr Ala AspVal Glu Glu Ala Ala Arg Lys Leu Ala Ala Ile Leu 100 105 110 Arg Glu GluArg Ala Asp Val Leu Thr Val Tyr Asp Glu Lys Gly Thr 115 120 125 Tyr GlyAsp Pro Asp His Ile Gln Val His Arg Val Gly Thr Arg Ala 130 135 140 AlaGlu Leu Ala Gly Thr Ala Lys Val Phe Gln Ser Thr Ile Asn Arg 145 150 155160 Glu His Ile Lys Ala Asn Gln Arg Val Leu Ala Glu Gln Ala Gly Val 165170 175 Asp Leu Pro Ala Gly Pro Asp Phe Gly Thr Pro Glu Ala Glu Leu Thr180 185 190 Cys Arg Val Asp Val Ser Ala Tyr Thr Glu Tyr Lys Arg Lys AlaLeu 195 200 205 Leu Ala His Ala Ser Gln Ile Thr Pro Gln Ser Thr Leu PheThr Asp 210 215 220 Leu Pro Glu Asp Thr Phe Arg Thr Met Phe Gly Thr GluTrp Phe Ile 225 230 235 240 Arg Ala Gly Gln Gly Pro Gly Ile Thr Glu ThrAsp Leu Met Ala 245 250 255 15 303 PRT Mycobacterium tuberculosis 15 MetSer Glu Thr Pro Arg Leu Leu Phe Val His Ala His Pro Asp Asp 1 5 10 15Glu Ser Leu Ser Asn Gly Ala Thr Ile Ala His Tyr Thr Ser Arg Gly 20 25 30Ala Gln Val His Val Val Thr Cys Thr Leu Gly Glu Glu Gly Glu Val 35 40 45Ile Gly Asp Arg Trp Ala Gln Leu Thr Ala Asp His Ala Asp Gln Leu 50 55 60Gly Gly Tyr Arg Ile Gly Glu Leu Thr Ala Ala Leu Arg Ala Leu Gly 65 70 7580 Val Ser Ala Pro Ile Tyr Leu Gly Gly Ala Gly Arg Trp Arg Asp Ser 85 9095 Gly Met Ala Gly Thr Asp Gln Arg Ser Gln Arg Arg Phe Val Asp Ala 100105 110 Asp Pro Arg Gln Thr Val Gly Ala Leu Val Ala Ile Ile Arg Glu Leu115 120 125 Arg Pro His Val Val Val Thr Tyr Asp Pro Asn Gly Gly Tyr GlyHis 130 135 140 Pro Asp His Val His Thr His Thr Val Thr Thr Ala Ala ValAla Ala 145 150 155 160 Ala Gly Val Gly Ser Gly Thr Ala Asp His Pro GlyAsp Pro Trp Thr 165 170 175 Val Pro Lys Phe Tyr Trp Thr Val Leu Gly LeuSer Ala Leu Ile Ser 180 185 190 Gly Ala Arg Ala Leu Val Pro Asp Asp LeuArg Pro Glu Trp Val Leu 195 200 205 Pro Arg Ala Asp Glu Ile Ala Phe GlyTyr Ser Asp Asp Gly Ile Asp 210 215 220 Ala Val Val Glu Ala Asp Glu GlnAla Arg Ala Ala Lys Val Ala Ala 225 230 235 240 Leu Ala Ala His Ala ThrGln Val Val Val Gly Pro Thr Gly Arg Ala 245 250 255 Ala Ala Leu Ser AsnAsn Leu Ala Leu Pro Ile Leu Ala Asp Glu His 260 265 270 Tyr Val Leu AlaGly Gly Ser Ala Gly Ala Arg Asp Glu Arg Gly Trp 275 280 285 Glu Thr AspLeu Leu Ala Gly Leu Gly Phe Thr Ala Ser Gly Thr 290 295 300

That which is claimed is:
 1. A purified acyl glucosaminyl inositol amidase, characterized as having: a) an N-terminal region with an amino acid sequence with at least 80% sequence identity to SEQ ID NO:2, b) four domains of conservation, wherein three of the domains contain conserved histidine residues, and c) amidase activity against acyl glucosaminyl inositol amides.
 2. A purified amidase of claim 1, wherein the amidase has amide hydrolase activity.
 3. A purified amidase of claim 1, wherein the acyl glucosaminyl inositol amide is a mycothiol-derived S-conjugate.
 4. A purified amidase of claim 1, wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.
 5. A purified amidase of claim 1, wherein the amidase is derived from an actinomycetes.
 6. A purified amidase of claim 1, wherein the amidase is a mycothiol S-conjugate amidase.
 7. A purified amidase of claim 5, wherein the amidase is derived from M. smegmatis and the N-terminal region has the amino acid sequence as set forth in SEQ ID NO.
 2. 8. A purified amidase of claim 5, wherein the amidase is derived from M. tuberculosis.
 9. A purified amidase of claim 5, wherein the amidase is derived from M. leprae.
 10. A purified amidase of claim 5, wherein the amidase is derived from M bovis.
 11. A purified amidase of claim 5, wherein the amidase is derived from M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum. M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, or M. avium.
 12. A purified amidase of claim 1, wherein the amidase is derived from an antibiotic-producing bacterium.
 13. A purified amidase of claim 12, wherein the bacterium is selected from the group consisting of Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei and Saccharopolyspora erythraea.
 14. A purified amidase of claim 1, wherein the amidase is encoded by a polynucleotide having a nucleic acid sequence as set forth in nucleic acid residues 34318-35184 of GenBank Accession No. gi2896719.
 15. A purified amidase of claim 1, wherein the amidase has an amino acid sequence as set forth in GenBank Accession No. CAA17198.
 16. A purified amidase of claim 1, wherein the amidase has an amino acid sequence as set forth in SEQ ID NO:1.
 17. A purified amidase of claim 1, wherein the amidase is encoded by a polynucleotide comprising a nucleic acid sequence as set forth in SEQ ID NO:6.
 18. An antibody, or functional fragment thereof, that binds specifically to an amidase of claim
 1. 19. An isolated polynucleotide that encodes an amidase of claim
 1. 20. A vector containing a polynucleotide that encodes an amidase of claim
 1. 21. A cell transformed with a vector of claim
 20. 22. A method for identifying an inhibitor of acyl glucosaminyl inositol amidase, said method comprising: a) contacting a candidate compound with an amidase of claim 1 or a polynucleotide encoding the amidase in the presence of an GlcN-Ins-containing amide under suitable conditions and b) determining the presence or absence of breakdown products of the amide indicative of amide hydrolase activity, wherein the substantial absence of the amide hydrolase activity is indicative of a candidate compound that inhibits activity of acyl glucosaminyl inositol amidase.
 23. The method of claim 22, wherein the amidase is an acyl glucosaminyl inositol amidase.
 24. The method of claim 22, wherein the amidase is a mycothiol-derived S-conjugate amide.
 25. The method of claim 22, wherein the breakdown product is 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins).
 26. The method of claim 22, wherein the breakdown product is a mercapturic acid.
 27. The method of claim 22, wherein the three domains in the amidase that contain conserved histidine residues have amino acid sequences selected from SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.
 28. The method of claim 22, wherein the amidase is produced in an actinomycete.
 29. The method of claim 28, wherein the actinomycete is M. smegmatis and the N-terminal region of the amidase has the amino acid sequence as set forth in SEQ ID NO:2.
 30. The method of claim 28, wherein the actinomycete is M. tuberculosis.
 31. The method of claim 28, wherein the actinomycete is M. leprae.
 32. The method of claim 28, wherein the actinomycete is M. bovis.
 33. The method of claim 28, wherein the actinomycete is M. intracellulare, M africanum, M. marinarum, M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, or M. avium.
 34. A method for increasing production of antibiotic by antibiotic-producing bacteria, said method comprising: contacting the antibiotic-producing bacteria with a compound that increases intracellular production by the bacteria in culture of an acyl glucosaminyl inositol amidase of claim 1; wherein the increase in production of the amidase increases the production of antibiotic by the bacteria by increasing resistance of the bacteria to the antibiotic.
 35. The method of claim 34, wherein the antibiotic-producing bacteria are actinomycetes.
 36. The method of claim 34, wherein candidate compound is a polypeptide, polynucleotide or small molecule.
 37. The method of claim 35 wherein the compound is a polynucleotide that encodes the amidase and the actinomycetes are transformed with the polynucleotide so as to express the amidase in culture.
 38. The method of claim 37, wherein the polynucleotide comprises a nucleic acid sequence as set forth in SEQ ID NO:6.
 39. The method of claim 37, wherein the polynucleotide encodes a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:1.
 40. The method of claim 35, wherein the actinomycetes are selected from the group consisting of Streptomyces lincolnensis, Amycolatopsis mediterranei, Amycolatopsis orientalis, Streptomyces lavendulae, Streptomyces coelicolor, Streptomyces rochei and Saccharopolyspora erythraea.
 41. The method of claim 34, wherein the three domains have amino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.
 42. The method of claim 34, wherein the three domains have amino acid sequences at least 80% identical to amino acid sequences selected from the group consisting of SEQ ID NOs:3, 4, 5, and any combination of two or more thereof.
 43. A method for decreasing the antibiotic-resistance of pathogenic acyl glucosaminyl inositol amidase-producing bacteria, said method comprising: introducing into the bacteria an inhibitor of acyl glucosaminyl inositol amidase activity, wherein the intracellular presence of the inhibitor decreases activity of the amidase, thereby decreasing the antibiotic-resistance of the bacteria as compared with untreated control bacteria.
 44. The method of claim 43, wherein the inhibitor inhibits intracellular production of the amidase.
 45. The method of claim 43, wherein the inhibitor inhibits intracellular amidase activity of the amidase.
 46. The method of claim 43, wherein the inhibitor is an anti-sense oligonucleotide complementary to a target region in a messenger RNA that encodes a polypeptide having an amino acid sequence segment with at least 80% sequence identity to the amino acid sequence of SEQ ID NOS:2, 3, 4 or 5, and conservative variations thereof.
 47. The method of claim 43, wherein the oligonucleotide hybridizes under intracellular conditions with a messenger RNA that encodes a polypeptide having an N-terminal amino acid sequence as set forth in SEQ ID NO:2.
 48. The method of claim 43, wherein the bacteria are actinomycetes.
 49. The method of claim 48, wherein the bacteria are pathogenic bacteria are M. smegmatis, M. tuberculosis, M. leprae, M. bovis, M. intracellulare, M. africanum, M. marinarum. M. chelonai, Corynebacterium diphtheria, Actinomyces israelii, or M. avium.
 50. The method of claim 43, wherein the amidase is a mycothiol S-conjugate amidase.
 51. The method of claim 50, wherein conjugate partner in the S-conjugate is an electrophile.
 52. The method of claim 51, wherein the electrophile is an alkyl or alkyloid group.
 53. The method of claim 43, wherein the S-conjugate amide is a mycothiol-derived S-conjugate amide.
 54. The method of claim 43, wherein the bacteria are actinomycetes.
 55. The method of claim 54, wherein the actinomycetes are M. smegmatis and the N-terminal region has the amino acid sequence as set forth in SEQ ID NO.
 2. 56. A method for detoxifying a toxic substance comprising contacting the toxic substance with bacteria transformed with a polynucleotide that encodes an amidase of claim 1 and expressing the amidase in order to detoxify the toxic substance.
 57. The method of claim 56, wherein the amidase is expressed under environmental conditions.
 58. The method of claim 57, wherein the environmental condition is a pollutant.
 59. The method of claim 58, wherein the pollutant includes a halogenated hydrocarbon, 1, 2 dibromoethane, 1,2 dichloroethane, perchloroethene, trichloroethene, isoprene, or vinyl chloride.
 60. A process for preparation of 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside (GlcN-Ins), said method comprising: contacting an N-acyl glucosaminyl inositol with an amidase of claim 1 under suitable conditions so as to hydrolyze the amide bond therein, and obtaining the GlcN-Ins.
 61. The process of claim 60, wherein the N-acyl glucosaminyl inositol is a mycothiol S-conjugate.
 62. The process of claim 61, wherein the mycothiol S-conjugate is the bimane derivative of mycothiol.
 63. The process of claim 61, wherein N-acetyl glucosamine inositol is the N-acyl glucosamine inositol.
 64. The process of claim 63, wherein the N-acetyl glucosamine inositol is N-acetyl- 1-D-myo-inosityl-2 amino-2 deoxy-α-D-glucopyranoside. 